Derivatives of cyclodepsipeptide PF 1022

ABSTRACT

Novel PF 1022 derivatives--cyclodepsipeptides represented by the below-described formula (I)--and acid addition salts thereof, which have been synthesized according to the present invention, have anthelmintic activities against various parasitic worms which are parasitic on human bodies, domestic animals and pet animals. They are therefore useful as anthelmintics for the prevention or treatment of parasitic infections. ##STR1## wherein R 1 , R 2 , R 3 , R 4 , Q, X, Y and Z have been defined herein.

TECHNICAL FIELD

The present invention relates to novel derivatives having acyclo-tetradepsipeptide skeletal structure in common to PF 1022, whichis a cyclodepsipeptide and has anthelmintic activities, and havingexcellent anthelmintic activities; and also to anthelmintic agentcontaining the derivatives. The novel PF 1022 derivatives according tothe present invention show excellent anthelmintic activities againstworms parasitic on animals and therefore are useful as anthelminticagent.

BACKGROUND ART

The PF 1022 substance is a cyclodepsipeptide which was found as a resultof a study on anthelmintic compounds against fowl roundworms seeJapanese Patent Application Laid-Open (Kokai) No. HEI 3-35796, EuropeanPatent Application Publication No. 0382173A2 and "J. Antibiotics", 45,692(1992)!. In addition, the PF 1022 substance is a fermentationproduct, which is produced by the culture of a filamentous fungus PF1022strain (deposited under FERM BP-2671 with National Institute ofBioscience and Human-Technology Agency in Tsukuba-shi under theprovisions of the Budapest Treaty) belonging to Agonomycetales and is acyclodepsipeptide represented by the following formula (A): ##STR2##

The PF 1022 substance is a depsipeptide which is formed ofL-N-methylleucine (CH₃)₂ --CH--CH₂ --CH(NH--CH₃)COOH!, D-lactic acid CH₃--CH(OH)COOH! and D-phenyllactic acid C₆ H₅ --CH₂ --CH(OH)COOH! viaester-bonds and amido-bonds and which can also be represented by thefollowing formula (A'):

    Cyclo-(L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-PhLac)(A')

wherein MeLeu is an N-methylleucine residue represented by the followingformula: ##STR3## Lac is a lactic acid residue represented by thefollowing formula: ##STR4## and PhLac is a phenyllactic acid residuerepresented by the following formula: ##STR5##

By the culture of the above filamentous fungus PF 1022 strain, the PF1022B substance of the following formula (B), the PF1022C substance ofthe formula (C), the PF 1022D substance of the formula (D) and the PF1022E substance of the formula (E) are produced in addition to the abovePF 1022 substance. They have anthelmintic activities and were isolatedby the present inventors see Japanese Patent Application No. HEI3-163085, now Japanese Patent Application Laid-Open (Kokai) No. HEI5-170749; but concerning PF 1022E, Japanese Patent Application No. HEI4-279094 (filed Oct. 19, 1992 but not yet laid open)!. ##STR6##

Incidentally, the PF 1022E substance is a novel compound which has notbeen reported yet.

The above-described PF 1022 substance and PF 1022B to E substances allpossess anthelmintic activities and have a marked structuralcharacteristic in that they have a large cyclo-tetradepsipeptidestructure as a basic skeleton, and that they have, as side chains, notonly four N-methyl groups and four isobutyl groups but also 0-3 methylgroup(s) and 1-4 benzyl group(s) and contain eight asymmetric carbonatoms in their molecules. In the skeletal cyclodepsipeptide structure ofthe group of these PF 1022 substances, a 24-membered ring is formed via4 ester bonds and 4 amide-bonds. This structure can be presumed to playan important role on the development of biological activities.

So-called helmintic infections cause serious damage to the human andanimal health and also to agriculture. There is a steady demand towardnovel and useful substances having anthelmintic activities and alsoadvantageous preparation processes for such anthelmintically activesubstances. Paying attention to such a demand, the present inventorsstudied with a view toward preparing and providing novel substancesrelated to the PF 1022 substance.

The PF 1022 substance is a fermentation product of the above-describedfilamentous fungus. Makoto Ohyama et al. proposed, as a process ofpreparing the PF 1022 substance by total synthesis, a process whichcomprises the steps shown in the following reaction route map (A) seeJapanese Patent Application No. HEI 4-131139 (filed: May 22, 1992) andJapanese Patent Application Laid-Open No. HEI 5-320148 (laid open: Dec.31 1993)!. ##STR7##

Incidentally, as one example of hitherto known totally syntheticprocesses for preparation of cyclodepsipeptides, the process reported in"Agric. Biol. Chem." 43(5), 1079-1083(1979), which is related to totalsynthesis of enniatin C, can be given.

DISCLOSURE OF THE INVENTION

With a view to providing novel cyclodepsipeptides which have acyclo-tetradepsipeptide skeletal structure in common to the PF 1022substance but exhibit anthelmintic activities superior to the PF 1022substance, the present inventors have conducted a variety of research.As a result, it has been found that a series of novel derivatives orrelated products of the PF 1022 substance can each be synthesized eitherby hydrogenating, in the presence of a rhodium catalyst under mildreaction conditions, one or more phenyl groups in the plural benzylgroups of the PF 1022 substance or the PF 1022B substance or the PF1022E substance so as to form cyclohexyl group(s), or by chemicallymodifying the phenyl groups through substitution reaction. It has alsobeen found that a series of novel derivatives of the PF 1022 substancecan each be prepared in accordance with total synthesis procedures byusing L-N-methylleucine (L-MeLeu) or L-leucine in combination with anα-hydroxycarboxylic acid, particularly a D- or L-lactic acid derivative,which may contain a substituent on its β-carbon atom, and thencondensing the carboxyl group of the leucine compound with theα-hydroxyl group of the lactic acid compound through an ester bond,condensing the carboxyl group of the resultant esterified product withthe amino group of the leucine compound through an amide bond,continuing further condensation of the condensation product as needed,thereby to synthesize a chain-like tetradepsipeptide, followed bycyclizing the tetradepsipeptide.

A series of novel PF 1022 derivatives synthesized as described above bythe present inventors can generically be represented by thebelow-described formula (I). It has been ascertained by animal teststhat these synthesized novel derivatives have useful anthelminticactivities.

In a first aspect of the present invention, there is thus provided acyclodepsipeptide, namely a PF 1022 derivative represented by thefollowing formula: ##STR8## wherein (i) R² and R⁴ are each acyclohexylmethyl group or benzyl group, R¹ and R³ are each a methylgroup or cyclohexylmethyl group or benzyl group, and X, Y, Z and Q areeach a methyl group, provided that at least one of R¹, R², R³ and R⁴ isa cyclohexylmethyl group, or (ii) R¹, R², R³ and R⁴ are each a linear orbranched alkyl group containing 1 to 11 carbon atoms and may be the sameor different from each other, and X, Y, Z and Q are each a methyl group,or (iii) R¹ and R³ are each a linear or branched alkyl group containing1 to 11 carbon atoms and may be the same or different from each other,and R² and R⁴ are each an unsubstituted benzyl group, and X, Y, Z and Qare each a methyl group, provided that R³ is not a methyl group when R¹is a methyl group, or (iv) R¹, R² and R³ are each a linear or branchedalkyl group containing 1 to 11 carbon atoms and may be the same ordifferent from each other and R⁴ is a benzyl group bearing or notbearing substituent(s) on the phenyl nucleus of the benzyl group, and X,Y, Z are Q are each a methyl group, or (v) both of R¹ and R³ are methylgroups while both of R² and R⁴ are benzyl groups, and at least one of X,Y, Z and Q is hydrogen but the remainders thereof are all methyl groups,or (vi) R¹, R³, X, Y, Z and Q are all methyl groups, R² is a benzylgroup bearing or not bearing substituent(s) on the phenyl nucleus of thebenzyl group and R⁴ is a benzyl group bearing substituent(s) on thephenyl nucleus of the benzyl group.

BEST EMBODIMENTS FOR WORKING THE INVENTION

The above-described novel PF 1022 derivative of the formula (I) embracestherein, as preferred embodiments, such hydrogenated derivatives of thePF 1022 substance, as represented by below-described formula (I-i-a);such hydrogenated derivatives of the PF 1022B substance, as representedby the below-described formula (I-i-b); cyclodepsipeptides of thebelow-described formula (I-ii), cyclodepsipeptides of thebelow-described formula (I-iii), cyclodepsipeptides of thebelow-described formula (I-iv), cyclodepsipeptides of thebelow-described formula (I-v), cyclodepsipeptides of the below-describedformula (I-vi-a) and cyclodepsipeptides of the below-described formula(I-vi-b).

(1) Hydrogenated derivatives of the PF 1022 substance, as represented bythe following formula: ##STR9## wherein R^(2a) and R^(4a) are each acyclohexylmethyl or benzyl group, provided that at least one of R^(2a)and R^(4a) is a cyclohexylmethyl group.

(2) Hydrogenated derivatives of the PF 1022B substance, as representedby the following formula: ##STR10## wherein R^(1b), R^(2b), R^(3b) andR^(4b) are each a cyclohexylmethyl group or benzyl group, provided thatat least one of R^(1b), R^(2b), R^(3b) and R^(4b) is a cyclohexylmethylgroup.

(3) Cyclodepsipeptides represented by the following formula: ##STR11##wherein R^(1c), R^(2c), R^(3c) and R^(4c) are each a linear or branchedalkyl group containing 1 to 11 carbon atoms, particularly an alkyl groupcontaining 1 to 6 carbon atoms and may be the same or different fromeach other.

(4) Cyclodepsipeptides represented by the following formula: ##STR12##wherein R^(1d) and R^(3d) are each a linear or branched alkyl groupcontaining 1 to 11 carbon atoms, particularly an alkyl group containing1 to 6 carbon atoms, and may be the same or different from each other,provided that R^(1d) and R^(3d) do not stand for methyl groups at thesame time.

(5) Cyclodepsipeptides represented by the following formula: ##STR13##wherein R^(1e), R^(2e) and R^(3e) are each a linear or branched alkylgroup containing 1 to 11 carbon atoms, particularly an alkyl groupcontaining 1 to 6 carbon atoms and may be the same or different fromeach other, and G, L and M denote independently a hydrogen or asubstituent, particularly a halo group, hydroxyl group, an alkoxy group,a lower alkenyloxy group, a phenyl-lower alkoxy group, analkylcarbonyloxy group, tetrahydropyranyloxy group or trityloxy group.

(6) Cyclodepsipeptides represented by the following formula: ##STR14##wherein at least one of X^(a), Y^(a), Z^(a) and Q^(a) is a hydrogen andthe reminders thereof are all methyl groups; and preferably either X^(a)and Z^(a) are methyl groups while Y^(a) and Q^(a) are hydrogens, orX^(a) and Z^(a) are hydrogens while Y^(a) and Q^(a) are methyl groups.

(7) Cyclodepsipeptides represented by the following formula: ##STR15##wherein G',L' and M' denote independently a substituent, particularly ahalo group, hydroxyl group, an alkoxy group, a lower alkenyloxy group, aphenyl-lower alkoxy group, an alkylcarbonyloxy group,tetrahydropyranyloxy group or trityloxy group.

(8) Cyclodepsipeptides represented by the following formula: ##STR16##wherein G', L' and M' denote independently a substituent, particularly ahalo group, hydroxyl group, an alkoxy group, a lower alkenyloxy group, aphenyl-lower alkyl group, an alkylcarbonyloxy group,tetrahydropyranyloxy group or trityloxy group.

Examples of the PF1022 derivative of the formula (I) according to thefirst aspect of the present invention are shown below in Table 1. InTable 1, each Example number corresponds to the Example number of thecorresponding compound whose Preparation Example will be describedhereinafter.

In Table 1, Me stands for a methyl group, Bn a benzyl group, ChxyMe acyclohexyl methyl group, i-Pr an isopropyl group, n-Bu an n-butyl group,sec-Bu a secondary butyl group and i-Bu an isobutyl group, respectively.

                                      TABLE 1                                     __________________________________________________________________________     ##STR17##                                                                    Example                                                                            Substance code                                                                       R.sup.1                                                                            R.sup.2                                                                             R.sup.3                                                                            R.sup.4   X Y Z Q                                 __________________________________________________________________________    1 & 2                                                                              PF1022-AHH                                                                           Me   ChxyMe                                                                              Me   Bn        Me                                                                              Me                                                                              Me                                                                              Me                                 1   PF1022-ADH                                                                           Me   ChxyMe                                                                              Me   ChxyMe    Me                                                                              Me                                                                              Me                                                                              Me                                 1   PF1022-BTH                                                                           ChxyMe                                                                             ChxyMe                                                                              ChxyMe                                                                             ChxyMe    Me                                                                              Me                                                                              Me                                                                              Me                                 3   PF1022-002                                                                           Me   Me    Me   Me        Me                                                                              Me                                                                              Me                                                                              Me                                13   PF1022-209                                                                           Me   i-Pr  Me   i-Pr      Me                                                                              Me                                                                              Me                                                                              Me                                15   PF1022-217                                                                           Me   n-C.sub.16 H.sub.13                                                                 Me   n-C.sub.6 H.sub.13                                                                      Me                                                                              Me                                                                              Me                                                                              Me                                 9   PF1022-203                                                                           i-Pr Bn    i-Pr Bn        Me                                                                              Me                                                                              Me                                                                              Me                                10   PF1022-205                                                                           sec-Bu                                                                             Bn    sec-Bu                                                                             Bn        Me                                                                              Me                                                                              Me                                                                              Me                                11   PF1022-207                                                                           n-Bu Bn    n-Bu Bn        Me                                                                              Me                                                                              Me                                                                              Me                                12   PF1022-225                                                                           Me   Bn    n-Bu Bn        Me                                                                              Me                                                                              Me                                                                              Me                                14   PF1022-216                                                                           Me   n-C.sub.6 H.sub.13                                                                  Me   Bn        Me                                                                              Me                                                                              Me                                                                              Me                                16   PF1022-218                                                                           Me   Bn    Me   Bn        Me                                                                              H Me                                                                              H                                 17   PF1022-219                                                                           Me   Bn    Me   Bn        H Me                                                                              H Me                                 4   PF1022-003                                                                           Me   Bn    Me   i-Bu      Me                                                                              Me                                                                              Me                                                                              Me                                 6   PF1022E                                                                              Me   Bn    Me   p-hydroxy-                                                                              Me                                                                              Me                                                                              Me                                                                              Me                                                            benzyl                                             5   PF1022-005                                                                           Me   Bn    Me   p-methoxy-                                                                              Me                                                                              Me                                                                              Me                                                                              Me                                                            benzyl                                             7   PF1022-201                                                                           Me   p-benzyl-                                                                           Me   p-benzyl- Me                                                                              Me                                                                              Me                                                                              Me                                                 oxybenzyl  oxybenzyl                                          8   PF1022-202                                                                           Me   p-hydroxy-                                                                          Me   p-hydroxy-                                                                              Me                                                                              Me                                                                              Me                                                                              Me                                                 benzyl     benzyl                                            18   PF1022-215                                                                           Me   Bn    Me   p-t-butoxy-                                                                             Me                                                                              Me                                                                              Me                                                                              Me                                                            benzyl                                            19   PF1022-006                                                                           Me   Bn    Me   p-stearoyl-                                                                             Me                                                                              Me                                                                              Me                                                                              Me                                                            oxybenzyl                                         20   PF1022-011                                                                           Me   Bn    Me   3,5-di-iodo-4-                                                                          Me                                                                              Me                                                                              Me                                                                              Me                                                            hydroxybenzyl                                     21   PF1022-012                                                                           Me   Bn    Me   3,5-di-iodo-4-                                                                          Me                                                                              Me                                                                              Me                                                                              Me                                                            methoxybenzyl                                     22   PF1022-013                                                                           Me   Bn    Me   p-isobutyoxycarbo-                                                                      Me                                                                              Me                                                                              Me                                                                              Me                                                            nyloxybenzyl                                      23   PF1022-016                                                                           Me   Bn    Me   p-ethoxybenzyl                                                                          Me                                                                              Me                                                                              Me                                                                              Me                                24   PF1022-018                                                                           Me   Bn    Me   p-n-propoxybenzyl                                                                       Me                                                                              Me                                                                              Me                                                                              Me                                25   PF1022-019                                                                           Me   Bn    Me   p-isopropoxybenzyl                                                                      Me                                                                              Me                                                                              Me                                                                              Me                                26   PF1022-020                                                                           Me   Bn    Me   p-allyloxybenzyl                                                                        Me                                                                              Me                                                                              Me                                                                              Me                                27   PF1022-021                                                                           Me   Bn    Me   p-n-butoxybenzyl                                                                        Me                                                                              Me                                                                              Me                                                                              Me                                28   PF1022-022                                                                           Me   Bn    Me   p-benzyloxybenzyl                                                                       Me                                                                              Me                                                                              Me                                                                              Me                                29   PF1022-023                                                                           Me   Bn    Me   3,5-dichloro-4-                                                                         Me                                                                              Me                                                                              Me                                                                              Me                                                            hydroxybenzyl                                     30   PF1022-025                                                                           Me   Bn    Me   3,5-dibromo-4-                                                                          Me                                                                              Me                                                                              Me                                                                              Me                                                            hydroxybenzyl                                     31   PF1022-026                                                                           Me   Bn    Me   3,5-dibromo-4-                                                                          Me                                                                              Me                                                                              Me                                                                              Me                                                            methoxybenzyl                                     32   PF1022-029                                                                           Me   Bn    Me   p-n-octyloxy-                                                                           Me                                                                              Me                                                                              Me                                                                              Me                                                            benzyl                                            33   PF1022-224                                                                           Me   Bn    Me   p-tetrahydropyra-                                                                       Me                                                                              Me                                                                              Me                                                                              Me                                                            nyloxybenzyl                                      34   PF1022-223                                                                           Me   Bn    Me   p-trityloxybenzyl                                                                       Me                                                                              Me                                                                              Me                                                                              Me                                __________________________________________________________________________

In Table 1, the substances (substance code: PF1022-AHH, -ADH and -BTH)of Example 1 are examples of the derivative represented by the generalformula (I-i-a) or (I-i-b), while the substances (substance code:PF1022-209 and -217) of Example 13 and Example 15 are examples of thederivative represented by the general formula (I-ii). The substances(substance code: PF1022-203, -205, -207 and -225) of Examples 9, 10, 11and 12 are examples of the derivative represented by the general formula(I-iii). The substance (substance code: PF1022-216) of Example 14 is anexample of the derivative represented by the general formula (I-iv). Thesubstances (substance code: PF1022-218 and -219) of Examples 16 and 17are examples of the derivative represented by the general formula (I-v).The substances (substance code: PF1022-201 and -202) of Examples 7 and 8are examples of the derivative represented by the general formula(I-vi-a). Furthermore, the substances (substance code: PF1022-005,PF1022E, PF1022-215, -006, -011, -012, -013, -016, -018, -019, -020,-021, -022, -023, -025, -026, -029, -224 and -223) of Examples 5, 6 and18-34 are examples of the derivative represented by the general formula(I-vi-b).

Processes of preparing the PF1022 derivative of the general formula (I)according to the present invention will hereinafter be described.

(a) Preparation of the PF1022 substance or PF1022B substance byhydrogenation

Among the derivatives of the general formula (I) according to thepresent invention, hydrogenated derivatives or hydro-derivatives of thegeneral formula (I-i-a) or (I-i-b) can each be synthesized by using, asa starting material, the PF1022 substance or PF1022B substance asprepared by a fermentative method.

Chemical modification, particularly hydrogenation, of a benzene ringsuch as a phenyl group is generally regarded to be more difficult thanthe other reactions such as nitration or acylation which is the reactionby an electrophilic substitution. In general, hydrogenation made at hightemperature and under high pressure is widely made to reduce a phenylgroup into a cyclohexyl group. The PF1022 substance has a complexchemical structure as occurred naturally. It is presumed that if the PF1022 substance is subjected to hydrogenation under conventionalconditions at high temperature and under high pressure, decompositionreaction can also be involved by hydrogenolysis. Procedures ofhydrogenation under milder reaction conditions, that is, hydrogenationmade at normal temperature under normal pressure are desirable foreffecting the reduction of the phenyl groups in the PF 1022 substance.From such a viewpoint, the present inventors have proceeded with aninvestigation on the usability of various reducing catalysts. As aresult, it has been found that a rhodium catalyst is most suitable forthe hydrogenation of the phenyl groups in the side-chain benzyl groupsof the PF1022 substance to form cyclohexyl groups.

Examples of reducing catalysts, which are usable in the process forpreparing the invention derivative of the general formula (I-i-a) or(I-i-b) from the PF1022 substance by hydrogenation, include rhodium, andrhodium-carrier catalysts such as rhodium-carbon and rhodium-alumina,and cationic rhodium complexes such as tris(triphenylphosphine)rhodium.In practice, a rhodium-carbon catalyst is preferred. This hydrogenationprocess is able to minimize the hydrogen pressure and the extent ofheating upon the catalytic reduction, but elevated pressure and heatingto some extents are allow able so that the reaction time can beshortened and production of by-products can be suppressed. For a smoothprogress of the reaction, it is desired to dissolve the startingmaterials in an inert solvent such as methanol, ethanol or ethyl acetateand then to conduct the reaction while stirring the resultant solution.

Isolation of the target product (I-i-a) or (I-i-b) after the reactioncan be conducted by a well-known method, for example, filtration, columnchromatography or a fractional crystallization method using an inertsolvent.

(b) Preparation by a totally synthetic process

Derivatives of the general formula (I) according to the presentinvention can be prepared by a totally synthetic process, that is, byproviding the following compounds (1), (2), (3), (4), (5) and (6) andthen condensing them successively in proper combinations through anester-bond or an amide-bond.

The starting materials to be employed are as follows:

Compound (1): N-methyl-L-leucine (structural formula: (CH₃)₂ --CH--CH₂--CH(NH--CH₃)COOH, abbreviation: H-L-MeLeu-OH)

Compound (2): L-leucine (structural formula: (CH₃)₂ --CH--CH₂--CH(NH₂)COOH, abbreviation: H-L-Leu-OH)

Compound (3): D- or L-α-hydroxycarboxylic acid, preferably, D- orL-lactic acid or a lactic acid derivative having a desired substituentintroduced at its β-carbon atom, each being represented by the followingformula: ##STR18## will hereinafter be abbreviated as A¹. Compound (4):D- or L-α-hydroxycarboxylic acid represented by the following formula:##STR19## will hereinafter be abbreviated as A². Compound (5): D- orL-α-hydroxycarboxylic acid represented by the following formula:##STR20## will hereinafter be abbreviated as A³. Compound (6): D- orL-α-hydroxycarboxylic acid represented by the following formula:##STR21## will hereinafter be abbreviated as A⁴.

In the above formulas, R¹, R², R³ and R⁴ have the same meanings as R¹,R², R³ and R⁴ defined in connection with the general formula (I) givenhereinbefore.

In the first step of the totally synthetic process, the carboxyl groupof Compound (1) or (2) is reacted with the α-hydroxyl group of Compound(3), (4), (5) or (6). The following four Compounds (7)-(10) each havingan amino group at one end thereof and a carboxyl group at the other endthereof can therefore be prepared as primary condensates containing theester-linkage.

Compound (7): H-L-MeLeu(or Leu)-D-A¹ -OH

Compound (8): H-L-MeLeu(or Leu)-D-A² -OH

Compound (9): H-L-MeLeu(or Leu)-D-A³ -OH

Compound (10): H-L-MeLeu(or Leu)-D-A⁴ -OH

In the second and subsequent steps of the totally synthetic process, twoof Compounds (7)-(10) are condensed with each other in propercombinations via an amide-bond, whereby Compound (11), Compound (12),Compound (13) and Compound (14) are synthetically prepared in the orderas shown schematically in the below-described reaction route map B or C,or Compound (15) is synthetically obtained in the orders schematicallyshown in the hereinafter given reaction route map D. By cyclizing, viaan amide-bond, chain-like Compound (13) or Compound (15) which has anamino group at one end thereof and a carboxyl group at the other endthereof, a cyclic PF1022 derivative of the general formula (I) can beprepared.

Compound (11): H-L-MeLeu(or Leu)-D-A¹ -L-MeLeu(or Leu)-D-A² -OH

Compound (12): H-L-MeLeu(or Leu)-D-A¹ -L-MeLeu(or Leu)-D-A² -L-MeLeu(orLeu)-D-A³ -OH

Compound (13): H-L-MeLeu(or Leu)-D-A¹ -L-MeLeu(or Leu)-D-A² -L-MeLeu(orLeu)-D-A³ -L-MeLeu(or Leu)-D-A⁴ -OH

Compound (14): H-L-MeLeu(or Leu)-D-A³ -L-MeLeu(or Leu)-D-A⁴ -OH

Compound (15): H-(L-MeLeu(or Leu)-D-A¹ -L-MeLeu (or Leu)-D-A²)-OH##STR22##

For instance, when lactic acid is employed as each of Compounds (3),(4), (5) and (6), such a derivative of the general formula (I), whereR¹, R², R³ and R⁴ individually represent methyl group, can be obtained.When 2-hydroxyisovaleric acid is used as each of Compounds (3), (4), (5)and (6), such a derivative of the general formula (I), where R¹, R², R³and R⁴ individually represent an isopropyl group, can be obtained. When2-hydroxyhexanoic acid is employed as each of Compounds (3), (4), (5)and (6), such a derivative of the formula (I), where R¹, R²) R³ and R⁴individually represent a n-butyl group, can be obtained. When2-hydroxy-3-methylpentanoic acid is employed as each of Compounds (3),(4), (5) and (6), such a derivative of the formula (I) wherein R¹, R²,R³ and R⁴ individually represent a secondary butyl group can beobtained. When 2-hydroxy-4-methyl-n-valeric acid is employed as each ofCompounds (3), (4), (5) and (6), such a derivative of the formula (I)wherein R¹, R², R³ and R⁴ individually represent an isobutyl group canbe obtained. For example, when 2-hydroxyoctanoic acid is used asCompound (4), such a derivative of the formula (I) wherein R² representsn-C₆ H₁₃ can be obtained. When phenyllactic acid andp-hydroxyphenyllactic acid are employed as Compounds (4) and (6),respectively, such a derivative of the formula (I) wherein R² and R⁴represent benzyl and p-hydroxybenzyl groups, respectively, can beobtained.

In general, the a-hydroxycarboxylic acid which is Compound (4), (5) or(6) can be prepared by reacting a corresponding α-amino acid with sodiumnitrite to convert its amino group into a diazo group (--N₂), and thenconverting the diazo group to a hydroxyl group by acid treatment.

When the groups R¹ and R³ of the PF1022 derivative having the generalformula (I) are the same while the groups R² and R⁴ are the same, thatis to say, when the R¹ of the starting Compound (3) and the R³ ofCompound (5) are the same while the R² of the starting Compound (4) andthe R⁴ of Compound (6) are the same, it is only necessary to provideCompounds (3) and (4) as the starting α-hydroxycarboxylic acid for thepreparation of such a PF1022 derivative in accordance with the totallysynthetic process. Such target PF1022 derivative (I) (wherein R¹ ═R³,and also R² ═R⁴) can be prepared by using Leucine compound (1) or (2) incombination with Compound (3) or Compound (4), condensing them byesterification, preparing each of intermediates (7) and (8) with formingthe amide-bond, producing the chain-like Compound (15) via Compounds(11), and then cyclizing Compound (15), in accordance with the order asillustrated in the below-described reaction route map D. ##STR23##

In the PF1022 derivative of the general formula (I) and also in theα-hydroxycarboxylic acid compounds (3)-(6) as the starting materials,R¹, R², R³ and R⁴ can each be a C₁ -C₁₁ alkyl group. Specific examplesof such an alkyl group include methyl, ethyl, propyl, (specifically,n-propyl, iso-propyl), butyl (specifically, n-methyl, iso-butyl,sec-butyl, tert-butyl), pentyl (specifically, n-pentyl, iso-pentyl,sec-pentyl, 1,2-dimethylpropyl, neo-pentyl, 1-ethylpropyl,1,1-dimethylpropyl), hexyl, heptyl, octyl, nonyl and decyl groups.Preferred are lower (C₁ -C₆) alkyl groups.

When, in the compound of the general formula (1) or in Compound (3),(4), (5) or (6), R¹, R², R³ and R⁴ each represents a substituted orunsubstituted phenyl or benzyl group, on the other hand, specificexamples of such a group include phenyl; o-, m- and p-hydroxyphenyl; o-,m- and p-(C₁₋₁₀)alkylphenyl; o-, m- and p-(C₁₋₁₀)alkoxyphenyl; o-, m-,and p-halogeno(F, Cl, Br, I)phenyl. Other examples include benzyl; o-,m- and p-hydroxybenzyl; o-, m- and p-(C₁₋₁₀)alkylbenzyl; o-, m- andp-(C₁₋₁₀)alkoxybenzyl; and o-, m-, and p-halogeno(F, Cl, Br, I)benzyl.The number of the substituents on the phenyl nucleus of the benzyl groupcan be 1-4.

The process for preparation of the PF1022 derivative represented by thegeneral formula (I) in accordance with the totally synthetic processwill hereinafter be described with the above-described reaction routemap (B) or (C). In brief, in the first step, Compounds (7), (8), (9) and(10) are prepared by condensing Compound (1) or (2) with Compound (3);Compound (1) or (2) with Compound (4); Compound (1) or (2) with Compound(5); and Compound (1) or (2) with Compound (6) via an ester-bond,respectively.

At this time, the amino-protected leucine compound (1) or (2),andα-carboxyl-protected Compounds (3), (4), (5) and (6), that is,α-carboxyl-protected α-hydroxycarboxylic acids are employed. As acondensation method using the ester-bond, it is desired to conduct thecondensation with employing Compounds (3)-(6), each of which has itsα-hydroxyl group in the free form, in the presence of a condensationagent. When the carboxyl-protected Compound (3), (4), (5) or (6) is theD-isomer, its condensation is conducted in the presence of both DCC andan additive (a reagent, such as N-hydroxysuccinic acid imide,N-hydroxybenzotriazole or the like, which does not cause racemization inan ordinary peptide-forming reaction). When the carboxyl-protectedCompound (3), (4), (5) or (6) is the L-isomer, on the other hand,condensation is conducted while inverting the conformation of theα-hydroxyl group of Compound (3), (4), (5) or (6). The condensationmethod by the Cohen's reaction is desired because it does not causeracemization.

Furthermore, the amino-protected leucine compound (1) or (2) can becondensed, through an ester-bond, with a reactive derivative at the α-OHgroup of the carboxyl-protected Compound (3), (4), (5) and (6). In thismethod, when the α-carboxyl-protected compound (3), (4), (5) or (6) isthe D-isomer, it is desired that the compound has been made reactive bysubstituting the α-hydroxyl group by a chlorine atom, a bromine atom orthe like. When the compound is the L-isomer, it is desired that theα-hydroxyl group has been converted into a sulfonate ester such astosylate, methanesulfonate or the like.

According to the above condensation method, there is prepared theester-type compound (7), (8), (9) or (10) in which the carboxyl andamino groups are both protected. One of the protecting groups introducedin Compound (1) or (2) and Compounds (3)-(6), which are startingcompounds employed for the preparation of the above compounds (7)-(10),should be preferentially removable.

Examples of such a carboxyl-protecting group include those removableunder acid hydrolytic or reducing conditions, such as t-butyl, benzyl,p-methoxybenzyl, benzhydryl and trityl groups; and those removable underneutral conditions such as an allyl group.

Examples of the amino-protecting group include those removable underacid hydrolytic or reducing conditions such as benzyloxycarbonyl,t-butyloxycarbonyl, p-methoxybenzyloxycarbonyl and formyl groups; andthose removable under neutral conditions and commonly used in peptidechemistry, such as an aryloxycarbonyl group.

It is necessary to remove the carboxyl-protecting group and theamino-protecting group preferentially and independently from each of theprotected ester-type compounds (7), (8), (9) and (10) so obtained. Whenthe carboxyl-protecting group is removable under reducing conditions, itis necessary to select as the amino-protecting group an amino-protectinggroup removable under acid hydrolytic conditions. The converse case isalso feasible. When the amino-protecting group is removable underneutral conditions, for example, an aryloxycarbonyl group, it isnecessary to select as the carboxyl-protecting group acarboxyl-protecting group removable under acid hydrolytic conditions.The converse case is also feasible.

With regard to a method for removal of the carboxyl-protecting group oramino-protecting group, when the protecting group is removable underacid hydrolytic conditions, it is treated with trifluoroacetic acid,methanesulfonic acid, trifluoromethanesulfonic acid or the like.Treatment with trifluoroacetic acid is most preferred. When it isremovable under reducing conditions, treatment under catalytic reductionconditions using a palladium catalyst is desired. When the protectinggroup is removable under neutral conditions, for example, an aryl group,aryloxycarbonyl group or the like, it may be reacted with potassium2-ethylhexanoate in the presence of zero-valence palladium catalyst forthe deprotection.

In the next step, Compound (11) is prepared, as shown in the reactionroute map B, by condensing, the deprotected Compound (7) with thedeprotected Compound (8) through an amide-bond. As described above, itis possible to form an amide bond between the amino group of Compound(7) and the carboxyl group of Compound (8) or between the amino group ofCompound (8) and the carboxyl group of Compound (7) when thecondensation is done between Compounds (7) and (8). Compound (11)obtained by the condensation of Compounds (7) and (8) is condensedfurther with Compound (9). Here it is possible to form an amide-bondbetween the amino group of Compound (11) and the carboxyl group ofCompound (9) or between the amino group of Compound (9) and the carboxylgroup of Compound (11). Compound (12) obtained by the condensation ofCompounds (11) and (9) is then condensed with Compound (10). In thiscondensation step, it is possible to form an amide-bond between theamino group of Compound (12) and the carboxyl group of Compound (10) orbetween the amino group of Compound (10) and the carboxyl group ofCompound (12). As a result, Compound (13) can be prepared.

In the above respective condensation steps, the removal and freshintroduction of a protecting group are conducted appropriately as neededto obtain a desired amide-bond.

As described in the reaction route map C, Compound (9) can also becondensed with Compound (10). In this case, it is possible to form anamide-bond between the amino group of Compound (9) and the carboxylgroup of Compound (10) or between the amino group of Compound (10) andthe carboxyl group of Compound (9). Compound (11), which has beenobtained by the condensation of Compounds (7) and (8), can be condensedwith Compound (14), which has been obtained by the condensation ofCompound (9) and Compound (10). In this case, it is possible to form anamide-bond between the amino group of Compound (11) and the carboxylgroup of Compound (14) or between the amino group of Compound (14) andthe carboxyl group of Compound (11). As described in the reaction routemap D, Compound (15) can be prepared by bonding two molecules ofCompound (11) together through an amide-bond.

By the intramolecular ring closure of Compound (13) or Compound (15) soobtained, a derivative of the general formula (I) can be prepared. Thisring closure is effected by treating Compound (13) or Compound (15)using dicyclohexylcarbodiimide (DCC) or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and an additiveN-hydroxysuccinic acid imide (HOSU), 1-hydroxybenzotriazole (HOBt), orthe like! in combination.

Examples of solvents usable in the above ring-closure reaction includeether-solvents such as ether, tetrahydrofuran (THF) and 1,4-dioxane, andaprotic solvents such as N,N-dimethylformamide (DMF), acetonitrile andchloroform. Preferred is a mixed solvent of tetrahydrofuran andN,N-dimethylformamide. The ring-closure reaction can be carried out at0°-50° C., preferably 20°-30° C.

The totally synthetic process as described above is suited for thepreparation of the derivatives of the formulae (I-ii), (I-iii), (I-iv)and (I-v) among the derivatives of the general formula (I) according tothe present invention. (c) Preparation by introduction of substituent(s)into the PF1022 or PF1022E substance

Among the novel derivatives of the general formula (I), the derivativesof the formulas (I-vi-a) and (I-vi-b) can be prepared by introducing, inaccordance with known chemical methods, various substituents for thehydrogen(s) on the benzene ring (phenyl group) of the benzyl group, thatis, a side chain of the PF1022 substance or in the phenolic hydroxylgroup on the p-hydroxyphenylmethyl group (namely, benzyl group), thatis, a side chain of the PF1022 E substance refer to Japanese PatentApplication HEI 4-279094 (not yet laid open), and the synthetic Examplewhich will be described subsequently in Example 6)!.

Examples of the substituents, which may be introduced in the benzenering, namely, the phenyl nucleus of the benzyl group of the PF1022substance or in the phenolic hydroxyl group on the benzyl group of thePF1022E substance, include linear or branched alkyl groups, alkenylgroups, alkynyl groups, substituted or unsubstituted benzyl groups,diphenylmethyl group, triphenylmethyl group and acyl groups.Particularly preferred are alkanoyl, carbamoyl, methoxymethyl,methylthiomethyl and tetrahydropyranyl groups. Examples of thesubstituent(s) replaceable for the hydrogen(s) on the benzene ringinclude halogen atoms, as well as such substituents as replaceable forthe hydrogen(s) on an aromatic ring by ordinary electrophilicsubstitution.

The above substituent-introducing reaction can be performed in an inertsolvent by etherification, acylation, carbamoylation or the like. Theetherification can be conducted by a reaction with diazomethane ordiphenyldiazomethane, a reaction with isobutene or dihydropyrane in thepresence of an acid catalyst, or a reaction with an alkyl halide, analkenyl halide, an alkynyl halide, a benzyl halide, a substituted benzylhalide, or a triphenylmethyl chloride (namely, trityl chloride). While,the acylation can be conducted by a reaction with an acyl halide oralkyl chlorocarbonate in the presence of an organic base such astriethylamine or an inorganic base such as potassium carbonate. Most ofthe well-known reactions for the modification of a phenolic hydroxylgroup can be applied to as such. To the phenolic hydroxyl group at thepara position which is active to the electrophilic substitutingreaction, the halogenation or other well-known electrophilicsubstitutions can also be applied.

Derivatives of the general formula (I) according to the presentinvention, namely, derivatives of the formulae (I-i-a), (I-i-b), (I-ii),(I-iii), (I-iv), (I-v), (I-vi-a) and (I-vi-b) all have usefulanthelmintic activities and show a low acute toxicity to mammariananimals.

The novel PF1022 derivatives according to the present invention can beconverted to their acid addition salts by reacting them with apharmaceutically acceptable inorganic acid such as hydrochloric acid,sulfuric acid or phosphoric acid, or a pharmaceutically-acceptableorganic acid such as acetic acid, propionic acid, citric acid ormethanesulfonic acid. In addition, the PF1022 derivatives of the presentinvention or their salts can be formulated into anthelminticcompositions by mixing them with a pharmaceutically acceptable, solid orliquid carrier.

According to a second aspect of the present invention, therefore, thereis provided an anthelmintic composition characterized in that thecomposition comprises a novel cyclodepsipeptide represented by thegeneral formula (1) or its salt as an active ingredient.

The novel derivative of the general formula (I) according to the presentinvention or the composition containing the derivative can beadministered to animals orally or parenterally, for example, rectally.Through a proper preliminary test, the dose of the derivative can bedetermined depending on the kind of a parasite to be eliminated, thekind of a host animal to be treated and various other factors. As ageneral guideline, when orally administered, for example, for theelimination of fowl roundworms, oral administration of the compound ofthe formula (I) at a dose of 0.05 mg/kg or greater, preferably 0.2 mg to3 mg/kg is recognized to exhibit the anthelmintic action againstparasites.

The compound of the general formula (I) of the present invention can beformulated into an anthelmintic composition just in the same manner asfor the PF1022 substance, which is described in Japanese PatentApplication Laid-Open No. HEI 3-35796 or European Patent ApplicationPublication No. 0382173 A2.

Examples of the animal to which the PF1022 derivative of the formula (I)of this invention can be applied as an anthelmintic may include domesticanimals, poultry, experimental animals and pets, such as swine, cattle,horses, rabbits, sheep, goats, domestic fowls, ducks, turkeys, mice,white rats, guinea pigs, monkeys, dogs, cats and small birds.Illustrative parasites on these animals include parasites on cattle andsheep such as twisted stomachworms, stomachworms belonging to the genusOstertagia, small hairworms, nematodes belonging to the genus Cooperia,nodularworms belonging to the genus Oesophagostomum, amphisomes,intestinal tapeworms (Moniezia benedeni), lung worms and liver flukes;parasites on swine such as roundworms, whipworms and nodularworms;parasites on dogs such as roundworms, hookworms, whipworms and heartworms; parasites on cats such as roundworms and Spirometra mansoni; andparasites of chickens such as roundworms, hairworms and cecal worms. Thecompound of the present invention is also effective for the eliminationof parasites on human bodies such as roundworms, pinworms, hookworms(Ancylostoma duodenale, Ancylostoma ceylanicum, Necator americanus),oriental hairworms, strongyloides worms and whipworms.

The novel PF1022 derivative according to the present invention can beused for the treatment and prevention of parasitic infections. For thetreatment, the derivative may be administered orally or parenterally.Upon oral administration, a liquid preparation of the derivative may beforcedly administered using a stomach catheter or the like, oradministered after mixing it with daily feed or drinking water, oradministered in an ordinary dosage form suitable for oraladministration, such as tablets, capsules, pellets, boluses, powders orsoft capsules. Upon parenteral administration, it may be administeredsubcutaneously, intramuscularly, intravenously, intraperitoneally orthrough a similar route by injecting the derivative of the formula (I)prepared in the form of a water-insoluble preparation in peanut oil,soybean oil or the like or in the form of a water-soluble preparation inglycerol, polyethylene glycol, etc.

For the prevention of parasitic infections, it is a common practice toadminister the PF1022 derivative orally as a a mixture of it with dailyfeed. Although no limitation is imposed on the administration period inthe case of preventive purposes, it is, in most cases, sufficient toadminister it for about 2 months in the case of broiler chickens and forabout 5 months in the case of swine.

The dose of the PF1022 derivative according to the present invention mayvary depending on the kind of the animal to be treated, the kind of theparasite and the method of administration. For instance, when fowlroundworms are to be eliminated by oral administration of a liquidpreparation using a stomach catheter, it can be administered at 0.05mg/kg or more. For the preventive purposes, the derivative can be mixedwith feed at a concentration of 1 ppm or higher, preferably 5 to 10 ppmand administered continuously.

Furthermore, a solution or suspension of the PF1022 derivative of thepresent invention in a liquid carrier can be administered to animals bysubcutaneous or intramuscular injection, etc., For parenteraladministration, non-aqueous formulations using a vegetable oil such aspeanut oil or soybean oil are employed. Aqueous parenteral formulationswhich contain a water-soluble carrier such as glycerol or polyethyleneglycol can also be employed for parenteral administration. Theseformulations generally contain the compound of the present invention inan amount of 0.1 to 10 wt. %. Even when the PF1022 derivative of thepresent invention is orally administered to mice at the dose of 300mg/kg, normal body-weight gains are obtained without any abnormalities.This indicates the low toxicity of this substance.

The anthelmintic activities of the PF1022 derivative of the generalformula (I) according to this invention will be described by thefollowing Tests.

Test 1

Fowl Roundworm Eliminating Test

Fowls (three fowls per group), which had been artificially infected withfowl roundworms and whose infection therewith had been confirmed byscatoscopy, were used as experimental animals. Upon administration ofeach test substance, the test substance weighed in a dose (mg)accurately calculated on the basis of the body weight (kg) of each fowlwas suspended in carboxymethylcellulose-containing water, and theresulting suspension was administered orally as a single dose unit usinga stomach tube. After the administration, the worms eliminated from thefowls were counted daily. Seven days after the administration, the fowlwas sacrificed and autopsied and the worms remaining in its intestinaltract were counted. The percent elimination was calculated in accordancewith the following calculation equation: ##EQU1##

Test results are summarized in Table 2 shown below. Each test substanceis indicated by the corresponding substance code name shown above inTable 1.

                  TABLE 2                                                         ______________________________________                                        Test substance    Dose    % Elimina-                                          (Code name)       (mg/kg) tion                                                ______________________________________                                        PF1022 (Control)  0.5     50-70                                               PF1022 (Control)  1.0     60-86                                               PF1022 (Control)  2.0     100                                                 Not treated       0        0                                                  PF1022-AHH        5       62                                                  (Hexahydro derivative                                                         of Example 1)                                                                 PF1022-ADH        5       30                                                  (Dodecahydro derivative                                                       of Example 1)                                                                 PF1022-BTH        5       30                                                  (Tetracyclohexylmethyl                                                        derivative of Example 1)                                                      PF1022-022        5       84                                                  (Compound of Example 3)                                                       PF1022-003        10      76                                                  (Compound of Example 4)                                                       PF1022 E          0.5     70                                                  (Compound of Example 6)                                                       PF1022-005        0.5     73                                                  (Compound of Example 5)                                                                         1.0     89                                                                    2.0     100                                                 PF1022-016        0.5     55                                                                    1.0     61                                                                    2.0     100                                                 PF1022-020        1.0     54                                                                    2.0     100                                                 PF1022-021        1.0     41                                                                    2.0     90                                                  PF1022-022        1.0     40                                                                    2.0     93                                                  PF1022-215        1.0     37                                                                    2.0     98                                                  ______________________________________                                    

Test 2

In vivo Anthelmintic Activity Test on Nematodes

To each sheep which had artificially been infected with Trichostrongyluscolubriformis (hereinafter abbreviated as "T") and Haemonchus contortus(hereinafter abbreviated as "T"), respectively, a test substance weighedin a dose accurately calculated from the weight (mg) of the sheep wasorally administered in the form of a gelatin capsule.

The number of parasite's eggs excreted with feces from the sheep wascounted quantitatively before and after the administration so that thedegree of anthelmintic effects was determined. The anthelmintic effectsare evaluated by rated numeral 0, 1, 2 or 3. The anthelmintic effectsare rated "0" when there were no anthelmintic activities, "2" whenexcretion of parasite's eggs was observed, and "3" when excretion ofparasite's eggs stopped, that is, parasitic worms were removedcompletely.

Table 3 shows the results of the test on anthelmintic activities againstthe above-described two types sheep parasitic worms.

                  TABLE 3                                                         ______________________________________                                        Test        Parasitic   Dose    Degree                                        substance   worms       (mg/kg) of effects                                    ______________________________________                                        PF1022 (Control)                                                                          H           0.05    3                                             PF1022 (Control)                                                                          T           0.5     3                                             PF1022-201  H           0.25    3                                             PF1022-201  H           0.1     1                                             PF1022-215  H           0.25    3                                             PF1022-219  T           0.5     3                                             ______________________________________                                    

Test 3

Anthelmintic effects of certain PF1022 derivatives on a rat intestinalnematode were tested in accordance with the following method.

Sixteen male Wistar rats were divided into eight groups (two rats pergroup), and about 2,000 larval worms of Nippostrongylus brasillensiswere hypodermically inoculated per rat. Seven days after theinoculation, PF1022, PF1022E, PF1022-002, PF1022-003, PF1022-209,PF1022-218 and PF1022-219 were, as test substances, forcedlyadministered p.o. to the rats in the groups, respectively, in an amountof 10 mg/kg per rat. Upon administration, each test substance (8 mg) wasdissolved in 0.2 ml of dimethyl sulfoxide and then, the resultantsolution was diluted with distilled water to give a 2 ml suspension. Tendays after the inoculation, the rats were each subjected to autopsy andimaginal worms parasitic on the small intestines were counted.

As the test results, the average of remaining worms in each of the eightgroups and the percent effectiveness of the test substance in each groupas compared with that of the infected control group are shown in Table4. PF1022 showed percent effectiveness of 80%, but PF1022-003 andPF1022-209 showed the effectivenesses of 66.1% and 59.3%, respectively.

                  TABLE 4                                                         ______________________________________                                        Anthelmintic effects of each derivative on                                    N. brasillensis-infected rats                                                 Test                  Number of     % Effec-                                  substance   Dose      remaining worms                                                                             tive-                                     (code name) (mg/kg)   (average value ± SD)                                                                     ness                                      ______________________________________                                        PF 1022 (control      403 ±  32.5  80.2                                    PF 1022E    10        2175 ± 134.4 0                                       PF 1022-002 10        1976.5 ±                                                                             306.2 2.7                                     PF 1022-003 10        689 ±  48.1  66.1                                    PF 1022-209 10        827 ±  388.9 59.8                                    PF 1022-218 10        2400 ± 362.0 0                                       PF 1022-219 10        2084.5 ±                                                                             94.0  0                                       Control infected                                                                           0        2082 ± 297.0 --                                      ______________________________________                                    

Examples for preparing the novel derivatives of the general formula (I)according to this invention will be described specifically by thefollowing Examples, in which the abbreviations have the followingmeanings:

Bn: benzyl group

Boc: t-butoxycarbonyl group

BH: benzhydryl group (diphenylmethyl group)

Cbz: carbobenzoxy group

All: allyl group (1-propenyl group)

HP: tetrahydropyranyl group

Tr: triphenylmethyl group (trityl group)

TYR: tyrosine residue ##STR24## TYRA: p-hydroxyphenyllactic acid residue##STR25## Lac: lactic acid residue ##STR26## PhLac: phenyllactic acidresidue ##STR27## MeLeu: N-methylleucine residue ##STR28## Leu: Leucineresidue ##STR29## LEUA: 2-hydroxy-4-methyl-n-valeric acid residuesynthesized from leucine ##STR30## norLeu: norleucine residue ##STR31##norLEAU: 2-hydroxy-L-hexanoic acid residue ##STR32## isoLEAU:2-hydroxy-3-methyl-L-pentanoic acid residue ##STR33##

VALA: 2-hydroxyisovaleric acid residue ##STR34## OctA: 2-hydroxyoctanoicacid residue ##STR35## HOBt: 1-hydroxybenzotriazole BOP-Cl:N,N-bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride

DCC: dicyclohexylcarbodiimide

EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

TFA: trifluoroacetic acid

THF: tetrahydrofuran

DMF: N,N-dimethylformamide

DMSO: dimethylsulfoxide

NMM: N-methylmorpholine

DEAD: diethyl azodicarbonate

Illustrated in the following Examples 1-2 are preparation processes byhydrogenation.

EXAMPLE 1 Preparation of hydrogenation products of the PF1022 and PF1022B substances

To 2.20 g of a mixture of the PF1022 and PF1022 B substances, 70 ml ofethanol and 30 ml of ethyl acetate were added to dissolve the former inthe latter. A reaction mixture, which had been prepared by adding 1.0 gof 5% rhodium-carbon catalyst to the resulting solution, was stirredunder hydrogen gas at 1 atmospheric pressure so that the mixture wassubjected to catalytic reduction. Twenty two hours after the beginningof the stirring, that is, at the time when 222 ml of hydrogen had beenconsumed, the stirring was terminated and the catalyst was filtered offfrom the reaction mixture. The filtrate was concentrated under reducedpressure, whereby the residue was obtained in a colorless resin-likeform. The residue was dissolved in 10 l of a hexane-ethyl acetate (1:1)mixed solvent, followed by subjecting the resulting solution tochromatography on a silica gel column packed with 1 kg of silica gel("Silica Gel 601", product of Merck & CO.). Elution was then conductedusing as eluents 0.9 l of hexane-ethyl acetate (2:1), 1.0 l ofhexane-ethyl acetate (3:2) and 4.0 l of hexane-ethyl acetate (1:1), andthe following four fractions were collected as eluates.

Fraction 1 (0.7 l): Partial hydrogenation products of PF1022B werecontained therein;

Fraction 2 (0.9 l): Hydrogenation product of PF1022 was containedtherein;

Fraction 3 (0.9 l): Partially hydrogenation products of PF1022 werecontained therein; and

Fraction 4 (1.5 l): A mixture of partial hydrogenation products ofPF1022 and the starting material PF1022 was contained therein.

(1) Fraction 2 was concentrated under reduced pressure to obtain acolorless residue. To the residue, 10 ml of water were added, followedby stirring for 5 hours. The crystals so precipitated (448 mg) werecollected by filtration.

In an NMR spectrum of this substance (in CD₃ OD), no peak attributableto the aromatic hydrogens was observed. Further, in an EI mass spectrum,molecular ion peaks (M⁺) at 960, 905 and a fragment peak at 864 wereobserved. In its UV spectrum (methanol solution), the maximumabsorptions which had been observed at 263.6 nm and 257.6 nm for thePF1022 substance had disappeared. Said substance has thus been found tobe dodecahydro-PF1022, that is, a compound (substance code: PF1022-ADH)of the general formula (I-i-a) where cyclohexylmethyl groups are presentas R^(2a) and R^(4a), respectively.

Molecular formula: C₅₂ H₈₈ N₄ O₁₂

Specific rotation: α!_(D) -56.6° (c=0.15, methanol)

¹ H-NMR spectrum (in deutero-methanol), δ (ppm): 0.84-1.08 27H(m)1.17-2.08 38H(m) 1.42 3H(d,J=6.8) 2.87 3H(s) 2.97 3H(s) 3.07 3H(s) 3.173H(s) 4.81 1H(dd,J=4.1,10.4) 5.19 1H(q,J=6.8) 5.29 1H(dd,J=4.1,11.5)5.43-5.66 5H(m)

(2) To a colorless residue, which had been obtained by concentratingFraction 3 under reduced pressure, 20 ml of hexane and 0.5 ml ofmethanol were added. The resulting mixture was allowed to stand, wherebycolorless crystals precipitated. The crystals were collected byfiltration in a yield of 457 mg. The substance so obtained wascalculated to contain the five aromatic hydrogen atoms which areobserved from its NMR spectrum (in CD₃ OD). Further, on a UV spectrum,weakened maximum absorptions were observed at 263.6 and 257.6 nm. In anEI mass spectrum, on the other hand, the substance had molecular ionpeaks at 954(M⁺), 899 and 858. The substance has been found to behexahydro-PF1022 (substance code: PF1022-AHH), that is, a compound ofthe general the formula (I-i-a) where a benzyl group is present asR^(2a) and a cyclohexylmethyl group as R^(4a).

Molecular formula: C₅₂ H₈₂ N₄ O₁₂

Specific rotation: α!_(D) -79.6° (c=0.15, methanol)

¹ H-NMR spectrum (in deutero-methanol), δ (ppm): 0.75-1.07 27H(m)1.20-2.07 28H(m) 2.84, 2.89, 2.92, 2.95, 3.00, 3.07, 3.18 12H (each s,conformer) 3.05-3.22 2H(m) 4.75-4.82 1H(m) 5.14-5.32 2H(m) 5.36-5.855H(m) 7.25-7.34 5H(m)

(3) Fraction 1 was concentrated and the residue so obtained wasdissolved in a solvent. As described above, the resulting solution wassubjected to chromatography on a silica gel column, followed by elutionwith hexane-ethyl acetate (1:1). From eluate fractions, a hydrogenationproduct of the PF1022B substance was obtained. The solid so obtained wasrecognized to be a hydrogenation product (substance code: PF 1022-BTH)of the formula (I-i-b) in which the four benzyl groups of PF1022Bsubstance had been reduced into four cyclohexylmethyl groups.

EXAMPLE 2 Preparation of dodecahydro-PF1022

To 500 mg of the PF1022 substance, 30 ml of ethanol and 250 mg of 5%rhodium-carbon were added, followed by catalytic reduction for two daysunder hydrogen gas at 1 atm. After a stop of the consumption of hydrogenwas confirmed, the reaction was terminated. From the reaction mixture,the catalyst was removed using celite as a filtration assistant. Thefiltrate was concentrated under reduced pressure. The residue soobtained was added with water and small amounts of methanol andisopropyl ether, followed by stirring. The crystals so precipitated werecollected by filtration in a yield of 499 mg.

The substance so obtained was recognized as dodecahydro PF1022 (namely,PF1022-ADH). Incidentally, as a result of thin-layer chromatography onsilica gel (eluent: hexane-ethyl acetate, 1:1), no remainder of thestarting substance PF1022 or hexahydro PF1022 was recognized.

Preparation of the derivatives of the general formulae (I-ii)-(I-v) bythe totally synthetic process will be illustrated by the followingExamples 3-4 and Examples 6-17.

EXAMPLE 3 Synthesis of cyclo-(L-MeLeu-D-Lac-)4 (code: PF1022-002)

a. Synthesis of Boc-L-MeLeu-D-Lac-OH

In 10 ml of methanol, 1.065 g (2.54 mmol) of Boc-L-MeLeu-D-Lac-OBn weredissolved, followed by the addition of 128 mg of 10% Pd--C. Theresulting mixture was subjected to catalytic reduction under a hydrogenstream (for debenzylation). The reaction mixture obtained was filteredand then, the filtrate was concentrated, whereby 800 mg of the titlecompound were obtained (yield: 99%). The compound so obtained was fed tofor use in the next reaction without purification.

b. Synthesis of H-L-MeLeu-D-Lac-OBn

In 5 ml of methylene chloride, 1.065 g (2.68 mol) ofBoc-L-MeLeu-D-Lac-OBn were dissolved, followed by cooling to 50° C. Tothe resulting solution, 2 ml of TFA were added at the same temperature,followed by reaction at room temperature for 30 minutes (for removal ofBoc). The reaction mixture obtained was concentrated and the concentratewas dissolved in 50 ml of ethyl acetate. The solution so obtained waswashed with a saturated aqueous solution of sodium bicarbonate and asaturated aqueous solution of sodium chloride, followed by drying oversodium sulfate. The solvent was then distilled off from the driedsolution to give 822 mg of the title compound (Yield: 100%). Thecompound so obtained was fed to for use in the next reaction withoutpurification.

c. Boc-(-L-MeLeu-D-Lac-)₂ -OBn

In 10 ml of THF, 800 mg (2.54 mmol) of the compound synthesized inprocedure a) and 822 mg (2.68 mmol) of the compound synthesized inprocedure b) were dissolved. To the resulting solution, 542 mg of HOBt,0.3 ml of NMM and 0.86 g of DCC were added, followed by makingcondensation reaction at 4° C. for 2 days (for formation of amido-bond).Insoluble matter was filtered off from the resulting reaction mixture,and the filtrate was then added with 50 ml of ethyl acetate and 30 ml ofhexane. The resulting solution was washed with a 5% aqueous solution ofpotassium hydrogen sulfate, a saturated aqueous solution of sodiumbicarbonate and a saturated aqueous solution of sodium chloride,followed by drying over sodium sulfate. After the solvent was distilledoff from the dried solution, the residue was subjected to chromatographyon a silica gel column (chloroform:ethyl acetate, 50:1) for makingisolation and purification of the target compound. Thus, 1.20 g of thetitle compound were obtained (yield: 78%).

α!_(D) ²¹ : -44.7° (c=0.12, CHCl₃)

EI-MS m/s: 607 (M⁺)

¹ H-NMR(CDCl₃) δ: 0.88(d,3H,J=6.4 Hz), 0.92(d,3H, J=6.4 Hz),0.93(d,6H,J=6.4 Hz), 1.44 and 1.46(each s,9H), 1.51(d,3H,J=6.4 Hz),1.53(d,3H, J=6.4 Hz), 1.40-1.84(m,6H), 2.81,2.83,2.93 and 2.95(eachs,6H), 4.74 and 4.93(dd and t,J=4,11 Hz, and J=8 Hz), 5.10 (q,1H,J=6.4Hz), 5.12(q,1H, J=12.2 Hz), 5.20(d,1H, J=12.2 Hz), 5.25-5.36(m,2H),7.30-7.39(m,5H).

d. Synthesis of Boc-(-L-MeLeu-D-Lac-)₂ -OH

In a similar manner to the procedure a) of Example 3, 595 mg (0.98 mmol)of Boc-(-L-MeLeu-D-Lac-)₂ -OBn were subjected to catalytic reduction fordebenzylation, whereby 505 mg of the title compound were obtained(yield: 100%). The compound so obtained was fed to for use in the nextreaction without purification.

e. Synthesis of H-(-L-MeLeu-D-Lac-)₂ -OBn

In a similar manner to the procedure b) of Example 3, 634 mg (1.04 mmol)of Boc-(-L-MeLeu-D-Lac-)₂ -OBn were subjected to the Boc-removingreaction, whereby 526 mg of the title compound were obtained (yield:100%). The compound so obtained was provided for use in the nextreaction without purification.

f. Boc-(-L-MeLeu-D-Lac-)₄ -OBn

In 6 ml of THF, 505 mg (0.98 mmol) of the compound synthesized in theprocedure d) and also 526 mg (1.04 mmol) of the compound synthesized inthe procedure e) were dissolved. To the resulting solution, 204 mg ofHOBt, 0.11 ml of NMM and 0.33 g of DCC were added, followed by makingcondensation reaction at 4° C. for 24 hours. The reaction mixture soobtained was subjected to a similar treatment to the procedure c) ofExample 3, whereby 832 mg of the title compound were obtained (yield:83%).

α!_(D) ²¹ : -58.3° (c=0.28, CHCl₃)

EI-MS m/z: 1005 (M⁺)

¹ H-NMR(CDCl₃) δ: 0.86-1.03(10d,24H,J=6.4 and 6.7 Hz), 1.45 and1.46(each s,9H), 1.38-1.58(m,16H), 1.64-1.85(m,8H), 2.83-3.11(each s,12H), 4.45-4.56 and 4.74 (m and dd, 1H,J=4.1 and 11.1), 4.94(t,0.5H,J=8.1 Hz), 5.10(q,1H,J=7.1 Hz), 5.12(d,1H,J=12.2 Hz), 5.20(d,1H,J=12.2Hz), 5.13-5.40(m,5.5H), 7.30-7.39(m,5H).

g. Synthesis of cyclo-(-L-MeLeu-D-Lac-)₄

In a similar manner to the procedure b) of Example 3, 813 mg (0.89 mmol)of Boc-(-L-MeLeu-D-Lac-)₄ -OBn were deprotected by reaction with TFA.The reaction mixture so obtained was post-treated similarly and thecrude product so obtained was subjected to catalytic reduction andpost-treatment similarly to procedure d) of Example 3.

In 200 ml of THF, the amino acid derivative, H-(L-MeLeu-D-Lac-)₄ -OH soobtained was dissolved, followed by the addition of 0.55 g of HOBt and0.18 ml of NMM. The resulting mixture was added to a suspension of 0.60g of potassium chloride, 1.55 g of cesium chloride and 1.56 g ofEDCI.HCl in DMF (200 ml)--THF (400 ml), followed by reaction for 5 daysto effect the ring-closing reaction.

The resulting reaction mixture was added with 150 ml of ethyl acetate,followed by washing with 80 ml of water, 80 ml of a saturated aqueoussolution of sodium bicarbonate, 80 ml of a 5% aqueous solution ofpotassium hydrogen sulfate and 80 ml of a saturated aqueous solution ofsodium chloride and drying over sodium sulfate. The solvent was removedfrom the dried solution. The residue was subjected to chromatography ona silica gel column (chloroform:ethyl acetate=5:1→1:1) for the isolationand purification of the target compound, whereby 559 mg of the titlecompound were obtained (yield: 86%).

α!_(D) ²¹ : -68.2° (c=0.15, methanol)

m.p. 168°-170° C.

FAB-MS m/z: 797(M⁺)

¹ H-NMR(CDCl₃) δ: 0.85 and 0.95(each d, 6H,J=6.6 Hz), 0.89 and 0.98(eachd, 6H, J=6.8 Hz), 0.91 and 0.98(each d, 6H, J=6.8 Hz), 1.01 and1.07(each d, 6H,J=6.6 Hz), 0.99(d,3H,J=6.8 Hz), 1.36-1.51(m, 3H,J=6.4Hz), 1.55-1.65(m,1H), 1.42(d,3H,J=6.8 Hz), 1.44(d,3H,J=6.8),1.45(d,3H,J=6.8 Hz), 1.67-1.99(m,8H), 2.85(s,3H), 2.96(s,3H),3.07(s,3H), 3.16(s,3H), 4.78(dd,1H,J=4.3 and 11.1 Hz), 5.19(q,1H,J=6.8Hz), 5.29(dd,1H,J=5.6 and 10.4 Hz), 5.44(dd,1H,J=5.4 and 11.1 Hz),5.48(dd,1H,J=5.8 and 10.0 Hz), 5.58(q,1H,J=6.8 Hz), 5.64(q,1H,J=6.8 Hz),5.69(q,1H, J=6.8 Hz)

EXAMPLE 4 Synthesis ofcyclo-(-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-LEUA-)(Code: PF1022-003)

a. Synthesis of Boc-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In 15 ml of THF, 1.85 g (3 mmol) of Boc-L-MeLeu-D-PhLac-OH synthesizedin a similar manner to the procedure a) of Example 3 and also 1.016 g(3.3 mmol) of H-L-MeLeu-D-Lac-OBn synthesized in the procedure b) ofExample 3 were dissolved, followed by the addition of 1.5 ml ofpyridine, 535 mg (3.6 mmol) of HOBt and 817 mg (3.6 mmol) of DCC underice cooling. The resulting mixture was reacted for 15 hours, whereby thereactants were condensed by an amide-bond. Insoluble matter was removedfrom the reaction mixture, followed by post-treatment as in theprocedure c) of Example 3. Subsequent to the removal of the solvent fromthe solution, the residue obtained was subjected to chromatography on asilica gel column (toluene:ethyl acetate 10:1→5:1) for isolation andpurification, whereby 1.37 g of the title compound were obtained (yield:67%). The compound so obtained was fed to for use in the next reactionwithout further purification.

b. Synthesis of H-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In a similar manner to the procedure e) of Example 3, 1.15 g of thetitle compound were obtained (yield: 98%) from 1.37 g (2 mmol) of theprotected compound obtained above in the procedure a). The compound wasfed to for use in the next reaction without further purification.

c. Synthesis of Boc-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In a similar manner to the procedure a) of Example 4, 1.15 g (1.97 mmol)of H-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn synthesized in the procedure b)of Example 4 and 720 mg (1.97 mmol) of Boc-L-MeLeu-D-Lac-OH synthesizedin the procedure a) of Example 3 were condensed with each other by theamide-bond, whereby 1.30 g of the title compound were obtained (yield:75%). The compound was fed to for use in the next reaction withoutfurther purification.

d. Synthesis of H-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In a similar manner to the procedure e) of Example 3, 1.30 g (1.47 mmol)of the protected compound obtained above in procedure c) were treatedfor removal of Boc therefrom, whereby 1.28 g of the title compound wereobtained. The compound was fed to for use in the next reaction withoutfurther purification.

e. Synthesis ofBoc-L-MeLeu-D-LEUA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In a similar manner to the procedure a) of Example 4, 1.28 g (1.47 mmol)of the crude product, H-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBnsynthesized above in the procedure d) of Example 4 and 590 mg (1.64mmol) of Boc-L-MeLeu-D-LEUA-OH synthesized similarly to the procedure b)of Example 3 were condensed with each other, whereby 1.2 g of the titlecompound were obtained (yield: 73%). The compound was fed to for use inthe next reaction without further purification.

f. Synthesis ofcyclo-(-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-LEUA-)

In a similar manner to the procedure g) of Example 3, 1.2 g (1.07 mmol)of the compound synthesized in the procedure e) of Example 4 weresubjected to the deprotection and ring-closing reaction, whereby 433 mgof the title compound were obtained (yield: 44%).

α!_(D) ²¹ : -66.2° (c=0.15, methanol)

FAB-MS m/s: 915(M⁺)

¹ H-NMR(CD₃ OD) δ: 0.81 and 0.85(each d,6H,J=6.4 Hz), 0.86 and 0.95(eachd,6H,J=6.6 Hz), 0.89 and 0.98(each d,6H,J=6.8 Hz), 0.91 and 0.98(eachd,6H,J=6.8 Hz), 1.01 and 1.07(each d,6H,J=6.6 Hz), 0.99(d,3H, J=6.8 Hz),1.36-1.65(m,6H), 1.44(d,3H,J=6.8 Hz), 1.45(d,3H,J=6.8 Hz),1.67-99(m,8H), 2.85(s,3H), 2.96(s,3H), 3.07(s,2H), 3.16(s,3H),4.78(dd,1H,J=4.3 and 11.1 Hz), 5.19(q,1H,J=6.8 Hz), 5.29(dd,1H,J=5.6 and10.4 Hz), 5.44(dd,1H,J=5.4 and 11.1 Hz), 5.48(dd, 1H,J=5.8 and 10.0 Hz),5.58(q,1H,J=6.8 Hz), 5.64(q, 1H,J=6.8 Hz), 5.69(q,1H,J=6.8 Hz),7.23-7.34(5H,m).

EXAMPLE 5 Synthesis ofcyclo-(-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OMe)(Code: PF1022-005)

In 3 ml of THF, 99.2 mg (0.103 mmol) of PF1022 E substance, which canalso be expressed as"cyclo-(-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA)",were dissolved, followed by ice cooling. Under a nitrogen stream, theresulting solution was added with 0.02 ml (0.32 mmol) of methyl iodideand 9 mg (60% in oil, 0.23 mmol) of sodium hydride were added, followedby reaction for 40 minutes (for O-methylation). The reaction mixtureobtained was added with 20 ml of ethyl acetate, followed by washing with10 ml of a saturated aqueous solution of sodium chloride and drying overmagnesium sulfate. After removal of the solvent from the solution, theresidue obtained was subjected to preparative TLC (chloroform:ethylacetate=3:1) for making isolation and purification, whereby 88.4 mg ofthe title compound were obtained (yield: 88%).

α!_(D) ²¹ : -104° (c=0.13, methanol)

m.p. 103°-105° C. (recrystallized from MeOH-H₂ O-AcOEt)

FAB-MS m/z: 979(M⁺)

¹ H-NMR(CD₃ OD) δ: 0.78-1.05(each d,27H,J=6.4-7.0 Hz), 1.38(d,3H,J=7.0Hz), 1.3-1.5(m,4H), 1.5-1.9(m,8H), 2.81(s,3H), 2.88(s,3H), 2.90(s,3H),2.99(s,3H), 3.08(dd,1H,J=8.0 and 13.2 Hz), 3.09(dd,1H,J=7.8 and 13.2Hz), 3.17(dd,1H,J=7.3 and 13.2 Hz), 3.18(dd,1H, J=7.2 and 13.2 Hz),3.30(s,3H), 4.78(dd,1H,J=4.3 and 11.1 Hz), 5.19(q,1H,J=6.8 Hz),5.29(dd,1H,J=5.6 and 10.4 Hz), 5.44(dd,1H,J=5.4 and 11.1 Hz),5.48(dd,1H, J=5.8 and 10.0 Hz), 5.58(q,1H,J=6.8 Hz), 5.64(q,1H, J=6.8Hz), 5.69(q,1H,J=6.8 Hz), 6.80(d,2H,J=8.3 Hz), 7.20(d,2H, J=8.3 Hz),7.24-7.34(5H,m).

EXAMPLE 6

Synthesis of the PF1022 E substance, that is, PF1022 E which may also beexpressed as"cyclo-(-L-Meleu-D-Lac-L-MeLeu-D-TYRA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac)"

a.Boc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OBn

In 14 ml of THF were dissolved 1.40 g ofBoc-L-Meleu-D-Lac-L-Meleu-D-TYRA(OBn)-OH, 1.16 g ofH-L-Meleu-D-Lac-L-Meleu-D-PhLac-OBn and 247 mg of HOBT. The resultingsolution was added with 410 mg of DCC under ice cooling, followed bystirring overnight at room temperature (for the condensation reaction).After the precipitate so obtained was removed by filtration, thefiltrate was concentrated. The residue was added with 50 ml of ethylacetate. The resulting mixture was washed successively with a 5%solution of sodium sulfite, a saturated aqueous solution of sodiumbicarbonate and a saturated aqueous solution of sodium chloride and thendried over anhydrous magnesium sulfate, followed by filtration. Thefiltrate was concentrated and the residue was separated and purified bychromatography on a silica gel column (toluene:ethyl acetate=5:1),whereby 1.16 g of the title compound were obtained as a colorless oil(yield: 46.0%).

b. H-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OBn

In 11 ml of dichloromethane were dissolvedBoc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OBn(1.10 g). The resulting solution was added with 4 ml of TFA under icecooling, followed by stirring at room temperature for one hour. Thereaction mixture obtained was added with a small quantity of toluene andwas then concentrated. Ethyl acetate (50 ml) was added to theconcentrate, followed by washing successively with a saturated aqueoussolution of sodium bicarbonate and water and drying over anhydrousmagnesium sulfate. After filtration, the filtrate was concentrated andthe residue was fed to as such, for use in the next reaction.

c. H-L-MeLeu-D-Lac-L-MeLeu-D-TYRA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OH

In a mixed solution of 10 ml of methanol and 1 ml of water, 1.05 g ofH-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OBnwere dissolved. In a nitrogen atmosphere, the resulting solution wasadded with 100 mg of 10% Pd--C, followed by catalytic hydrogenation withhydrogen at room temperature under normal pressure for 5 hours. Thecatalyst was filtered off with using Hyflo Super Cel, and the filtratewas concentrated. The residue was fed to as such for use in the nextreaction.

d. Cyclo-(-L-MeLeu-D-Lac-L-MeLeu-D-TYRA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac)(namely, PF1022 E substance)

In a liquid mixture of 800 mg of THF and 240 ml of DMF, 477 mg oflithium chloride, 840 mg of potassium chloride, 610 mg of sodiumchloride, 1.75 g of cesium chloride and 4.1 g of EDCI.HCL were added. Tothe resulting mixture, a solution of 1.01 g ofH-L-MeLeu-D-Lac-L-MeLeu-D-TYRA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OH, 720 mgof HOBt and 0.24 ml of NMM in 120 ml of THF was added, followed bystirring overnight. After the solvents were distilled off, 450 ml ofethyl acetate and 220 ml of water were added to the resulting residue.The resulting mixture was allowed to separate into two layers. Theorganic layer so obtained was washed successively with a saturatedaqueous solution of sodium bicarbonate, a 5% solution of sodium sulfiteand a saturated aqueous solution of sodium chloride, followed by dryingover anhydrous magnesium sulfate and filtering the organic solution.

The filtrate was then concentrated. The residue so obtained was purifiedby chromatography on a silica gel column (chloroform:ethyl acetate=3:1)and then by reversed phase chromatography on a silylized silica,gelcolumn (CH₃ CN--H₂ O=85:15), whereby 324 mg of the title compound wereobtained as white powder (yield: 33%).

α!_(D) ²⁵ : -100° (c=1.0, MeOH)

¹ H-NMR(CD₃ OD) δ: 0.78-1.00(m,24H,δ-Me(MeLeu)), 1.04,1.05,1.38,1.39(each d, total 6H, β-Me(Lac)),1.28-1.90(m,12H,β-CH₂,τ-H(MeLeu)), 2.82-3.00(m,12H,NMe),2.93-3.20(m,4H,β-CH₂, (TYRA, PhLac)),4.76-5.81(m,8H,α-H), each 2H,J=8.4, aromatic(TYRA)), 7.24

MS(EI): M⁺ =964

EXAMPLE 7 Synthesis of cyclo-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ (code:PF1022-201)

a. Synthesis of O-benzyloxy-L-phenyllactic acid (H-L-TYRA(OBn)-OH,namely ##STR36##

In a mixed solution of 75 ml of 1,4-dioxane and 50 ml of water, 5.46 gof O-benzyl-L-tyrosine (H-L-TYR(OBn)-OH) were suspended. To theresulting suspension, 25 ml of 2.4N hydrochloric acid were added underice cooling to dissolve the latter in the former. The resulting solutionwas added with an aqueous solution of 4.14 g of sodium nitrite, and thenwith 75 ml of 1,4-dioxane, 15 ml of water and 10 ml of 2N aqueoushydrochloric acid, followed by reaction for 30 minutes (fordiazotization of the amino group of tyrosine). The reaction mixtureobtained was added with an aqueous solution of 1.38 g of sodium nitriteand 10 ml of 2N hydrochloric acid, followed by reaction at roomtemperature for 2 hours (for conversion of the diazo group into ahydroxyl group).

The resulting reaction mixture was added with 200 ml of ethyl acetate.The resulting mixture was allowed to separate into two layers. The waterlayer so obtained was extracted again with ethyl acetate. The ethylacetate extract was combined with the organic layer, followed by washingtwice with 50 ml portions of a 30% aqueous solution of sodium chloride,drying over anhydrous magnesium sulfate and then concentrating underreduced pressure, whereby 1.33 g of the title compound were obtained(yield: 24.3%).

¹ H-NMR(DMSO-d₆) δ=2.78(ddd,2H,J=0,4,0.8,1.4,4.4,β-CH₂),4.08(q,1H,J=0.4,0.8,α-H), 5.06(s,2H,CH₂ Ph), 7.01(d×2,4H,J=0.8,C₆ H₄),7.41(m,5H,Ph)

b. Synthesis of H-L-TYRA(OBn)-OK (potassium O-benzyl-L-phenyllactate)

In a mixed solution of 5.5 ml of methanol and 7.65 ml of chloroform,1.10 g of H-L-TYRA(OBn)-OH were dissolved under heating, followed by theaddition of a solution (1 g/10 ml) of potassium 2-ethylhexanoate inethyl acetate. When a precipitate started to appear, 15 ml of ethylacetate were added further to the resulting solution, followed bystirring for 17 hours. The precipitate so obtained was collected byfiltration, followed by washing with ethyl acetate and drying underreduced pressure, whereby 950 mg of the title compound were obtained(yield: 76.6%).

c: H-L-TYRA(OBn)-O-All (allyl O-benzyl-L-phenyl-lactate)

In 15 ml of DMF, 2.5 g of H-L-TYRA(OBn)-OK and 0.34 g of sodiumbicarbonate were dissolved under ice cooling. To the resulting solution,0.91 ml of allyl iodide was added and they were reacted at the sametemperature for 12 hours. The reaction mixture was added with 75 ml ofethyl acetate, followed by washing once with 50 ml of water and twicewith 50 ml portions of a 30% aqueous solution of sodium chloride, dryingover anhydrous magnesium sulfate and then concentrating. The residue soobtained was purified by chromatography on a silica gel column(toluene:ethyl acetate=6:1), whereby 2.15 g of the title compound wereobtained (yield: 86.0%).

¹ H-NMR(CDCl₃): δ=3.00(ddd,2H,J=0.5,0.7,1.4,4.1,β-CH₂),4.43(q,1H,J=0.4,0.7,α-H), 4.64(m,2H,CH₂ (allyl)), 5.03(s,2H,CH₂ Ph),5.32(m,2H,CH₂ (Allyl)), 5.90(m,1H,CH(allyl)), 7.02(d×2,4H,J=0.9,C₆ H₄),7.37(m,5H,Ph)

d. Synthesis of Boc-L-MeLeu-D-TYRA(OBn)-O-All

In 8 ml of THF, 1.71 g of Boc-L-MeLeu-OH and 1.75 g oftriphenylphosphine were dissolved. The resulting solution was addeddropwise with a solution of 2.08 g of H-L-TYRA(OBn)-O-All and 1.09 ml ofDEAD in 4 ml of THF under ice cooling, followed by reaction at the sametemperature for 16 hours (for making condensation via an ester-bond).The reaction mixture obtained was concentrated and the residue waspurified by chromatography on a silica gel column (toluene:ethylacetate=20:1), whereby 3.51 g of the title compound were obtained(yield: 98.0%).

¹ H-NMR(CDCl₃): δ=0.91(s×2,6H,δ-CH₃ (Me-Leu)), 1.47(s×2,9H,CH₃ (Boc)),1.38-1.64(m,3H,β-CH₂,γ-H(MeLeu), 2.72(d,3H,J=1.24,N--CH₃),3.10(m,2H,β-CH₂ (TYRA)), 4.59(m,2H,CH₂ (allyl)),4.7-5.0(m,1H,α-H(MeLeu)), 5.03(s,2H,CH₂ Ph), 5.17-5.32(m,3H,α-H(TYRA),CH₂ (allyl)), 5.83(m,1H,CH(allyl)), 7.02(d×2,4H,J=0.8,C₆ H₄),7.36(m,5H,Ph)

e. Synthesis of H-L-MeLeu-D-TYRA(OBn)-O-All

In 15 ml of TFA, 3.49 g of Boc-L-MeLeu-D-TYRA(OBn)-O-All were dissolved,followed by reaction at 20° C. for 20 minutes (for removal of Boc). Theresultant reaction solution was concentrated. Toluene was add to theconcentrate, and TFA was removed azeotropically. The residue wasdissolved in 50 ml of ethyl acetate. The resulting solution was washedsuccessively with a 7% aqueous solution of sodium bicarbonate, water anda 30% aqueous solution of sodium chloride, each, in an amount of 50 ml,dried over anhydrous magnesium sulfate and concentrated under reducedpressure, whereby 2.68 g of the title compound were obtained (94.4%).The product so obtained was fed to as such for use in the next reaction.

f. Synthesis of Boc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-O-All

In a mixed solution of 20 ml of THF and 2 ml of pyridine, 1.98 g ofH-L-MeLeu-D-TYRA(OBn)-O-All and 2.47 g of Boc-L-MeLeu-D-Lac-OH weredissolved. The resulting solution was added with 0.96 g of HOBt and 1.13g of DCC under ice cooling, followed by stirring at the same temperaturefor 16.5 hours. After the precipitate was filtered off, the filtrate wascon centrated. The residue so obtained was purified by chromatography ona silica gel column (toluene:ethyl acetate=6:1), whereby 2.58 g of thetitle compound were obtained (yield: 77.4%).

g. Synthesis of H-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-O-All

In 6.5 ml of methylene chloride, 1.29 g ofBoc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-O-All were dissolved. TFA (6.45ml) was added dropwise to the resulting solution under ice cooling,followed by stirring for 20 minutes. The reaction mixture wasconcentrated under reduced pressure. Toluene was added to theconcentrate, and TFA was removed azeotropically. The residue wasdissolved in ethyl acetate. The resulting solution was washedsuccessively with a 7% aqueous solution of sodium bicarbonate, water anda 30% aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then, concentrated, whereby 1.05 g of the titlecompound were obtained (yield: 94.0%). The product was fed to as suchfor use in the next reaction

h. Synthesis of Boc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-OH

In 6.5 ml of methylene chloride, 1.29 g ofBoc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-O-All were dissolved, followed bythe addition of 44 mg of triphenylphosphine. To the resulting solution,8.7 mg of tetrakis(triphenylphosphine)palladium were added in a nitrogenatmosphere to dissolve the latter in the former. The resulting solutionwas added with 0.87 ml of 2N potassium 2-ethylhexanoate, followed bystirring for 5 minutes. The reaction mixture was concentrated and theresidue was dissolved in ethyl acetate. The resulting solution wasacidified with hydrochloric acid. The acidified solution was washedsuccessively with water and a 30% aqueous solution of sodium chloride,dried over anhydrous sodium sulfate and then concentrated, whereby thetitle compound were obtained. The product so obtained was fed to assuch, for use in the next reaction.

i. Synthesis of Boc-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ -O-All

In 16 ml of tetrahydrofuran, 1.51 g ofBoc-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-OH, 1.04 g ofH-L-MeLeu-D-Lac-L-MeLeu-D-TYR(OBn)-O-All, 260 mg of HOBt and 0.27 ml oftriethylamine were dissolved. The resulting solution was added with 428mg of DCC under ice cooling, followed by stirring for 13 hours (formaking condensation). The precipitate obtained was filtered off and thefiltrate was then concentrated. The residue was dissolved in ethylacetate. The resulting solution was washed successively with 5%potassium bisulfate, a 7% aqueous solution of sodium bicarbonate and a20% aqueous solution of sodium chloride, dried over anhydrous magnesiumsulfate and then concentrated under reduced pressure. The residue waspurified by chromatography on a silica gel column (toluene:ethylacetate=4:1), whereby 950 mg of the title compound were obtained (yield:44.4%).

j. Synthesis of Boc-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ -O-BH

In 4.75 ml of methylene chloride, 950 mg of Boc-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ -O-All were dissolved. The resulting solution wasadded with 18 mg of triphenylphosphine, followed by the addition of 4 mgof tetrakis(triphenylphosphine)palladium in a nitrogen atmosphere. Afterthey were dissolved completely, 0.36 ml of 2N potassium 2-ethylhexanoatewas added to the resulting solution, followed by stirring for 5 minutes.

The reaction mixture obtained was acidified with 2N hydrochloric acid,followed by washing successively with water and a 30% aqueous solutionof sodium chloride and drying over anhydrous sodium sulfate. Subsequentto the removal of the anhydrous sodium sulfate by filtration, a solutionof 196 mg of diphenyldiazomethane in ethyl acetate was added to theresulting filtrate. The resulting mixture was concentrated and theresidue was purified by chromatography on a silica gel column(benzene:ethyl acetate=5:1), whereby 1.08 g of the title compound wereobtained (yield: 100%).

k. Synthesis of H-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ -OH

In 5.4 ml of methylene chloride, 900 mg ofBoc-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ -O-BH were dissolved. Theresulting solution was added dropwise with 2.7 ml of TFA under icecooling, followed by reaction at the same temperature for 1.5 hours. Thereaction mixture was concentrated under reduced pressure. Toluene wasadded to the concentrate and TFA was removed azeotropically, whereby1.12 g of the title compound were obtained. The compound so obtained wasfed to as such for use in the next reaction.

l. Synthesis of cyclo-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ (code:PF1022-201)

To a mixture of 263 mg of lithium chloride, 362 mg of sodium chloride,463 mg of potassium chloride, 1.04 g of cesium chloride, 1.19 g ofED-CI.HCl, 650 ml of THF and 190 ml of DMF, there was added a solutionof 734 mg of H-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ -OH, 419 mg of HOBtand 0.2 ml of NMM in 100 ml of THF. The resulting mixture was stirred atroom temperature for 36 hours.

The reaction mixture was concentrated. The residue was then dissolved in200 ml of ethyl acetate. The resulting solution was washed successivelywith water, a 7% aqueous solution of sodium bicarbonate, a 5% aqueoussolution of potassium hydrogen sulfate and a 30% aqueous solution ofsodium chloride, each in 200 ml-portions, dried over anhydrous sodiumsulfate and then concentrated under reduced pressure. The residue soobtained was purified by chromatography on a silica gel column(chloroform:ethyl acetate=3:1), whereby 436 mg of the title compoundwere obtained (yield: 60.5%).

α!_(D) ²⁰ : -84.5° (c=1.0, Methanol)

¹ H-NMR(CD₃ OD) δ: 0.82-1.05(m,27H,δ-CH₃ (MeLeu),β-CH₃ (Lac)),1.38(d,3H,J=0.7,β-CH₃ (Lac)), 1.40-1.90(m,12H,β-CH₂,γ-H(MeLeu)),2.82,2.86,2.91, 2.99(each s,12H,N--CH₃), 2.90-3.20(m,4H,β-CH₂ (TYRA)),5.05(s,4H,CH₂ Ph), 4.70-5.82(m,8H,α-H(MeLeu,TYRA, Lac)),7.08(d×2,8H,J=0.9,C₆ H₄), 7.35(m,10H,Ph)

EXAMPLE 8 Synthesis of cyclo-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA)₂ (code:PF1022-202)

In 3 ml of methanol, 271 mg ofcyclo-(L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn))₂ were dissolved. To theresulting solution, 27 mg of 10% Pd/C were added to conduct catalytichydrogenation. About 30 minutes after the initiation of the catalytichydrogenation, a white precipitate appeared. The precipitate so formedwere dissolved in 0.75ml of THF and a small quantity of acetic acid asadded thereto. The resulting solution was-again subjected to thehydrogenation for 22 hours. The reaction mixture was added with 27 mg of10% Pd/C and the hydrogenation was continued for further 30 hours. Afterthe removal of the catalyst by filtration, the filtrate was concentratedunder reduced pressure. The residue was purified by chromatography on asilica gel column (chloroform:ethyl acetate=2:1→1:1), whereby 142 mg ofthe title compound were obtained as white powder (yield: 63.0%).

α!_(D) ²⁰ : -110° (c=0.1, Methanol)

¹ H-NMR(CD₃ OD): δ=0.83-1.06(m,27H,δ-CH₃ (MeLeu),β-CH₃ (Lac)),1.38(d,3H,J=0.6,β-CH₃ (Lac)), 1.39-1.95(m,12H,β-CH₂,γ-H(MeLeu)),2.82,2.86,2.92, 2.99(each s,12H,N--CH₃), 2.70-3.15(m,4H,β-CH₂ (TYRA)),4.70-5.80(m,8H,α-H(MeLeu,TYRA,Lac)), 6.91(d×2,8H, J=0.9,C₆ H₄)

EXAMPLE 9 Synthesis of cyclo(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ (code:PF1022-203)

a. L-2-Hydroxyisovaleric acid

To a liquid mixture of 170 ml of water, 170 ml of acetic acid and 40 mlof 1,4-dioxane, were added 23.4 g (0.2 mol) of L-valine, followed byheating to 40° C. to dissolve the valine in, said liquid. To theresulting solution, a solution of 41.4 g of sodium nitrite in 50 ml ofwater was added dropwise. The resultant mixture was stirred and reactedat room temperature for 3 hours. Under ice cooling, the reaction mixtureobtained was added to a liquid mixture of 300 ml of a saturated aqueoussolution of sodium chloride and 750 ml of ethyl acetate. The resultingmixture was allowed to separate into two layers. The water layer soobtained was extracted four times with 100 ml-portions of ethyl acetate.The combined organic layers were dried over anhydrous magnesium sulfate.The magnesium sulfate was filtered off and the filtrate was concentratedunder reduced pressure, whereby the title compound was obtained.

b. Diphenylmethyl L-2-hydroxyisovalerate (H-L-VALA-O-BH)

Ethyl acetate (300 ml) was added to the compound obtained in theprocedure a) to dissolve the latter in the former. To the resultingsolution, a solution of diphenyldiazomethane in ethyl acetate (38.8g/400 ml) was added dropwise, followed by stirring overnight at roomtemperature. Acetic acid (30 ml) was added to the reaction mixture,followed by washing thrice with 300-ml portions of a saturated aqueoussolution of sodium bicarbonate. The organic layer was dried overanhydrous magnesium sulfate, followed by filtration. The filtrate wasconcentrated under reduced pressure and the residue so obtained was thenpurified by chromatography on a silica gel column (toluene:ethylacetate=20:1), whereby 28.3 g of the title compound were obtained as anoil (yield: 49.7%).

1H-NMR(CD₃ OD): δ=0.76(d,3H,J=7.0,Me), 1.02(d,3H, J=7.0,Me),2.67(d,J=6.2,OH), 6.96(s,1H,CHPh₂), 7.25-7.40(m,10H,Ph)

c. Diphenylmethyl L-2-(p-tosyloxy)isovalerate

In 270 ml of dichloromethane, 26.8 g (94 mmol) of the compound obtainedin the procedure b) were dissolved. To the resulting solution, 57.2 g oftosyl chloride and 32.4 ml of pyridine were added, followed by stirringovernight at room temperature. Dichloromethane and water (each 250 ml)were added to the reaction mixture. The resulting mixture was allowed toseparate into two layers. The organic layer was washed successively witha 2% aqueous solution of sodium bicarbonate, 200 ml of water and 200 mlof water and then dried over anhydrous magnesium sulfate. The magnesiumsulfate was removed from the organic layer by filtration. The filtratewas concentrated under reduced pressure, and the residue was thenpurified by chromatography on a silica gel column (toluene:ethylacetate=10:1), whereby 29.5 g of the title compound were obtained aspale yellow crystals (yield: 69.9%).

¹ H-NMR(CDCl₃): δ=2.37(s,3H,CH₃ C₆ H₄ SO₂), 6.79(s,1H, CHPh₂),7.18(d,2H,J=8.4,CH₃ C₆ H₄ So₂), 7.22-7.35 (m,10H,Ph),7.70(d,2H,J=8.4,CH₃ C₆ H₄ SO₂)

d. Boc-L-MeLeu-D-VALA-OBH

In 20 ml of DMSO, 4.90 g (20 mmol) of Boc-L-MeLeu-OH and 13.2 g of thecompound (tosylate) obtained above in the procedure c) were dissolvedunder heating at 50° C., followed by the gradual addition of 5.52 g ofpotassium carbonate. The resulting mixture was stirred and reacted at50° C. for 4.5 hours (for esterification). To the reaction mixture,ethyl acetate and water (each 50 ml) were added. The resulting mixturewas allowed to separate into two layers. The water layer so obtained wasextracted again with 50 ml of ethyl acetate. The combined organic layers(the extracts) were washed with a 10% aqueous solution of sodiumchloride and then dried over anhydrous magnesium sulfate. Afterfiltration, the filtrate was concentrated and the residue obtained wasthen purified by chromatography on a silica gel column (chloroform:ethylacetate=50:1), whereby 3.75 g of the title compound were obtained aspale yellow crystals (yield: 36.6%).

α!_(D) : -57.1° (c=0.15, CHCl₃)

¹ H-NMR(CDCl₃): δ=1.46(s,9H,t-Bu), 2.72(d,3H,NMe), 6.91(s,1H,CHPh₂),7.26-7.34(m,10H,Ph)

e. Boc-L-MeLeu-D-VALA-OH

In a liquid mixture of 30 ml of methanol and 3 ml of water, 3.0 g (5.86mmol) of the compound obtained above in the procedure d) were dissolved.The resulting solution was added with 300 mg of 10% Pd--C in a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen gas atroom temperature under 1 atom., for 5 hours (for removal of benzhydrylgroup, BH). The catalyst was filtered off with aid of Hgflo Super Celand the filtrate was concentrated. The concentrate was fed to as suchfor use in the next reaction.

f. H-L-MeLeu-D-PhLac-OBn

In 5 ml of dichloromethane, 4.51 g (9.33 mmol) ofBoc-L-MeLeu-D-PhLac-OBn were dissolved. The resulting solution was addedwith 4 ml of TFA under ice cooling, followed by stirring at roomtemperature for 3 hours. The reaction mixture was added with a smallquantity of toluene and concentrated. Ethyl acetate (75 ml) was thenadded to the concentrate. The resulting mixture was washed with asaturated aqueous solution of sodium bicarbonate and water, dried overanhydrous magnesium sulfate and then filtered. The filtrate wasconcentrated and the residue was fed as such for use in the nextreaction.

g. Boc-L-MeLeu-D-VALA-L-MeLeu-D-PhLac-OBn

In a liquid mixture of 30 ml of THF and 3 ml of pyridine, 2.8 g ofBoc-L-MeLeu-D-VALA-OH, 2.47 g of H-L-MeLeu-D-PhLac-OBn and 950 mg ofHOBt were dissolved. The resulting solution was added with 1.452 g ofDCC under ice cooling, followed by stirring overnight at roomtemperature. The precipitate was removed from the reaction mixture byfiltration and the filtrate was concentrated. Ethyl acetate (280 ml) wasthen added to the residue. The resulting mixture was washed successivelywith 140 ml of a 5% solution of sodium sulfite, 140 ml of a saturatedaqueous solution of sodium bicarbonate and 140 ml of a saturated aqueoussolution of sodium chloride, dried over anhydrous magnesium sulfate andthen filtered. The filtrate was concentrated. The concentrate obtainedwas then purified by chromatography on a silica gel column(toluene→toluene:ethyl acetate=10:1), whereby 3.20 g of the titlecompound were obtained as a colorless oil (yield: 74.2%).

α!_(D) : -38.5° (c=0.5, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.82-1.08(m,18H,δ-Me(MeLeu),γ-Me(VALA)),1.43,1.45(each s,9H,t-Bu), 2.85,2.86(each s,each 3H,NMe),7.16-7.36(m,10H,Ph)

h. H-L-MeLeu-D-VALA-L-MeLeu-D-PhLac-OBn

In 2 ml of dichloromethane, 1.1 g (1.74 mmol) ofBoc-L-MeLeu-D-VALA-L-MeLeu-D-PhLac-OBn were dissolved. The resultingsolution was added with 2 ml of TFA under ice cooling, followed bystirring at room temperature for 1.5 hours. The reaction mixture wasadded with a small quantity of toluene and concentrated. Ethyl acetate(50 ml) was then added to the concentrate. The resulting mixture waswashed with a saturated aqueous solution of sodium bicarbonate andwater, dried over anhydrous magnesium sulfate and then filtered. Thefiltrate was concentrated. The concentrate containing the title compoundwas fed to directly for use in the next reaction.

i. Boc-L-MeLeu-D-VALA-L-MeLeu-D-PhLac-OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 1.1 g (1.74mmol) of Boc-L-MeLeu-D-VALA-L-MeLeu-D-PhLac-OBn were dissolved. Theresulting solution was added with 110 mg of 10% Pd--C in a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen gas atroom temperature under normal pressure for 2 hours. The catalyst wasremoved by filtration with aid of Hyflo Super Cel and the filtrate wasconcentrated. The residue was provided, as was, for use in the nextreaction.

j. Boc-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)2-OBn

In 10 ml of THF, 990 mg of L-Boc-MeLeu-D-VALA-L-MeLeu-D-PhLac-OH, 987 mgof H-L-MeLeu-D-VALA-L-MeLeu-D-PhLac-OBn and 282 mg of HOBt weredissolved. The resulting solution was added with 431 mg of DCC under icecooling, followed by stirring overnight at room temperature (forcondensation reaction). The precipitate was filtered off and thefiltrate was concentrated. Ethyl acetate (30 ml) was added to theconcentrate. The resulting mixture was washed successively with a 5%solution of sodium sulfite, a saturated aqueous solution of sodiumbicarbonate and a saturated aqueous solution of sodium chloride, driedover anhydrous magnesium sulfate and then filtered. The filtrate wasconcentrated. The residue so obtained was then purified bychromatography on a silica gel column (toluene:ethyl acetate=5:1),whereby 1.38 g of the title compound were obtained (yield: 67%).

α!_(D) : -63.9° (c=0.1, CHCl₃)

k. H-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ -OBn

In 4 ml of dichloromethane, 1.20 g (1.02 mmol) ofBoc-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ -OBn were dissolved. The resultingsolution was added with 2 ml of TFA under ice cooling, followed bystirring at room temperature for one hour. The reaction mixture wasadded with a small quantity of toluene and concentrated. Ethyl acetate(30 ml) was then added to the concentrate. The resulting mixture waswashed successively with a saturated aqueous solution of sodiumbicarbonate and water, dried over anhydrous magnesium sulfate andfiltered. The filtrate was concentrated and the residue so obtained wasfed as such for use in the next reaction.

l. H-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ -OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 1.15 g ofH-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ -OBn were dissolved, followed by theaddition of 110 mg of 10% Pd--C in a nitrogen atmosphere. The resultingmixture was subjected to catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 2 hours. The catalyst was removedby filtration with aid of Hyflo Super Cel and the filtrate wasconcentrated. The residue was supplied as such for use in the nextreaction.

m. Cyclo-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ (code: PF1022-203)

To a liquid mixture of 680 ml of THF and 200 ml of DMF were added 390 mgof lithium chloride, 685 mg of potassium chloride, 537 mg of sodiumchloride, 1.55 g of cesium chloride and 1.76 g of EDCI.HCl. To theresulting mixture, a solution of 910 mg ofH-(L-MeLeu-D-VALA-L-MeLeu-D-PhLac)₂ -OH, 620 mg of HOBt and 0.2 ml ofNMM in 110 ml of THF was added, followed by stirring overnight at roomtemperature (for the ring-closing reaction).

After the solvents were distilled off from the reaction mixture, 230 mlof ethyl acetate and 110 ml of water were added to the residue. Theresulting mixture was allowed to separate into two layers. The organiclayer so obtained was washed successively with a saturated aqueoussolution of sodium bicarbonate, a 5% solution of sodium sulfite and asaturated aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then filtered. The filtrate was concentrated. Theresidue so obtained was purified by subjecting to chromatography on asilica gel column, whereby 645 mg of the title compound were obtained ascolorless powder (yield: 56.5%).

α!_(D) : -71.6° (c=0.2, MeOH)

¹ H-NMR(CDCl₃): δ=0.79-1.01(m,36H,δ-CH₃ (MeLeu),γ-CH₃ (VALA)),1.38(d,3H,J=0.6,β-CH₃ (Lac)),1.53-2.05(m,12H,β-CH₂,γ-H(MeLeu),β-H(VALA)),2.60,2.79,2.82,2.89,2.90,3.11(each s,12H,N--CH₃), 2.70-3.27(m,4H,β-CH₂(PhLac), 4.36-5.98 (m,8H,α-H(MeLeu,VALA,PhLac)), 7.24-7.28(m,10H,Ph)

MS(FD):M⁺ =1004

EXAMPLE 10 Synthesis of cyclo-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂(code: PF1022-205)

a. 2-Hydroxy-3-methyl-L-pentanoic acid (H-L-isoLEUA-OH, namely, CH₃ CH₂CH(CH₃)--CH(OH)COOH)

In a liquid mixture of 500 ml of 1N hydrochloric acid and 50 ml of1,4-dioxane, 25 g (0.2 mol) of L-isoleucine were dissolved under heatingat 40° C., followed by cooling to room temperature. The resultingsolution was added dropwise with an aqueous solution of sodium nitrite(39.5 g/50 ml), and they were stirred at room temperature for 5 hours.The resulting reaction mixture was added to an ice-cooled solutionmixture of 300 ml of a saturated aqueous solution of sodium chloride and750 ml of ethyl acetate. The resulting mixture was allowed to separateinto two layers. The water layer was extracted four times with 100 mlportions of ethyl acetate. The combined organic layers were dried overanhydrous magnesium sulfate. After the magnesium sulfate was filteredoff, the filtrate was concentrated under reduced pressure. The residuewas supplied to as such for use in the next reaction.

b. Benzhydryl 2-hydroxy-3-methyl-L-pentanoate (H-L-isoLEUA-OBH)

In 300 ml of ethyl acetate, 2-hydroxy-3-methyl-L-pentanoic acid obtainedin the above procedure was dissolved. A solution of diphenyldiazomethanein ethyl acetate (41.2 g/60 ml) was added dropwise to the resultingsolution, followed by stirring overnight at room temperature. Theresulting reaction mixture was added with 30 ml of acetic acid, followedby washing thrice with 300 ml portions of a saturated aqueous solutionof sodium bicarbonate. The organic layer was dried over anhydrousmagnesium sulfate and then filtered. The filtrate was concentrated underreduced pressure. The residue so obtained was purified by subjecting tochromatography on a silica gel column (toluene:ethyl acetate=20:1),whereby 9.97 g of the title compound were obtained as an oil (yield:17%).

¹ H-NMR(CDCl₃): δ=0.82(t,3H,J=7.3,δ-Me), 0.97(d,3H,J=6.6,γ-Me),1.19(m,2H,CH₂), 1.90(m,1H,β-H), 2.70(d,1H,J=5.90,OH), 4.2(m,1H,α-H),7.0(s,1H,CHPh₂), 7.26-7.40(m,10H,Ph)

c. Boc-L-MeLeu-D-isoLEUA-OBH

In 30 ml of THF were dissolved 5.25 g of triphenylphosphine and 5.97 g(20 mmol) of H-L-isoLEUA-OBH. A solution of 5.89 g of Boc-L-MeLeu-OH and3.78 ml of DEAD in 10 ml of THF was added dropwise to the resultingsolution, followed by stirring overnight at room temperature. Theprecipitate was filtered off and 300 ml of ethyl acetate were added tothe residue. The resulting mixture was washed successively with asaturated aqueous solution of sodium bicarbonate, a saturated aqueoussolution of sodium chloride and water, followed by drying over anhydrousmagnesium sulfate. After filtration, the filtrate was concentrated. Theresidue was purified by subjecting to chromatography on a silica gelcolumn (toluene:ethyl acetate=50:1→10:1), whereby 7.01 g of the titlecompound were obtained as an oil (yield: 66.7%).

¹ H-NMR(CDCl₃): δ=1.44(s,9H,t-Bu), 4.8,5.1(each m, 1H,α-H(MeLeu)),5.14(t,1H,α-H(isoLEUA)), 6.96(s,1H,CHPh₂), 7.26-7.37(m,10H,Ph)

α!_(D) =-13.8° (c=0.55, CHCl₃)

d. Boc-L-MeLeu-D-isoLEUA-OH

In a liquid mixture of 50 ml of methanol and 5 ml of water, 5.26 g (10mmol) of Boc-L-MeLeu-D-isoLEUA-OBH were dissolved. The resultingsolution was added with 530 mg of 10% Pd--C in a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 5 hours. The catalyst was filtered off withaid of Hyflo Super Cel, and the filtrate was concentrated. The residuewas supplied as such for use in the next reaction.

e. H-L-MeLeu-D-PhLac-OBn

In 2 ml of dichloromethane, 2.90 g (6.0 mmol) of Boc-L-MeLeu-D-PhLac-OBnwere dissolved. The resulting solution was added with 2 ml of TFA underice cooling, followed by stirring at room temperature for 3 hours. Thereaction mixture was added with a small quantity of toluene andconcentrated. Ethyl acetate (50 ml) was added to the concentrate. Theresulting mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate and water, dried over anhydrous magnesiumsulfate and then filtered. The filtrate was concentrated and the residueobtained was supplied to as such for use in the next reaction.

f. Boc-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OBn

In 30 ml of THF, 2.93 g of Boc-L-MeLeu-D-isoLEUA-OH, 2.14 g ofH-L-MeLeu-D-PhLac-OBn and 904 mg of HOBt were dissolved. The resultingsolution was added with 1.38 g of DCC under ice cooling, followed bystirring overnight at room temperature (for the condensation). After theprecipitate was filtered off from the reaction mixture, the filtrate wasconcentrated. To the residue, 100 ml of ethyl acetate were added. Theresulting mixture was washed successively with 100 ml of a 5% aqueoussolution of sodium sulfite, 100 ml of a saturated aqueous solution ofsodium bicarbonate and 100 ml of a saturated aqueous solution of sodiumchloride, dried over anhydrous magnesium sulfate and filtered. Thefiltrate was then concentrated. The residue as obtained was purified bysubjecting to chromatography on a silica gel column (toluene:ethylacetate=10:1), whereby 1.80 g of the title compound were obtained as acolorless oil (yield: 47.9%).

g. H-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OBn

In 2 ml of dichloromethane, 875 mg (1.2 mmol) ofBoc-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OBn were dissolved. The resultingsolution was added with 2 ml of TFA under ice cooling, followed bystirring at room temperature for 1.5 hours. The reaction mixture wasadded with a small quantity of toluene and concentrated. Ethyl acetate(30 ml) was added to the concentrate. The resulting mixture was washedsuccessively with a saturated aqueous solution of sodium bicarbonate andwater, dried over anhydrous magnesium sulfate and then filtered. Thefiltrate was concentrated and the residue obtained was fed as such foruse in the next reaction.

h. Boc-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 875 mg (1.2mmol) of Boc-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OBn were dissolved. Theresulting solution was added with 85 mg of 10% Pd/C under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 3 hours. The catalyst was filteredoff with aid of Hyflo Super Cel and the filtrate was concentrated. Theresidue obtained was supplied as such for use in the next reaction.

i. Boc-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ -OBn

In 10 ml of THF, 668 mg of Boc-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OH, 732mg of H-L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac-OBn and 194 mg of HOBt weredissolved. The resulting solution was added with 297 mg of DCC under icecooling, followed by stirring overnight at room temperature (for thecondensation). After the precipitate was filtered off, the filtrate wasconcentrated. Ethyl acetate (30 ml) was added to the residue. Theresulting mixture was washed successively with a 5% aqueous solution ofsodium sulfite, a saturated aqueous solution of sodium bicarbonate and asaturated aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and filtered. The filtrate was concentrated, followedby purifying the resultant residue by chromatography on a silica gelcolumn (toluene:ethyl acetate=10:1→5:1), whereby 555 mg of the titlecompound were obtained as a colorless oil (yield: 37.3%).

j. H-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ -OBn

In 2 ml of dichloromethane, 555 mg (0.45 mmol) ofBoc-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ -OBn were dissolved. Theresulting solution was added with 2 ml of TFA under ice cooling,followed by stirring at room temperature for 1 hour. The reactionmixture was added with a small quantity of toluene and concentrated.Ethyl acetate (30 ml) was added to the concentrate. The resultingmixture was washed successively with a saturated aqueous solution ofsodium bicarbonate and water, dried over anhydrous magnesium sulfate andfiltered. The filtrate was then concentrated. The residue obtained wassupplied as such for use in the next reaction.

k. H-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ -OH

In a liquid mixture of 5 ml of methanol and 0.5 ml of water, 506 mg ofH-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ -OBn were dissolved. Theresulting solution was added with 50 mg of 10% Pd--C under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 2 hours. The catalyst was filteredoff with aid of Hyflo Super Cel and the filtrate was concentrated. Theresidue obtained was supplied as such for use in the next reaction.

l. Cyclo-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ (Code: PF1022-205)

To a liquid mixture of 400 ml of THF and 120 ml of DMF were added 216 mgof lithium chloride, 380 mg of potassium chloride, 298 mg of sodiumchloride, 859 mg of cesium chloride and 977 mg of EDCI.HCl. Theresulting mixture was added at room temperature with a solution, whichhad been obtained in advance by dissolving 529 mg ofH-(L-MeLeu-D-isoLEUA-L-MeLeu-D-PhLac)₂ -OH, 345 mg of HOBt and 0.11 mlof NMM in 60 ml of THF, followed by stirring overnight (for thering-closing reaction). After the solvent was distilled off, the residuewas added with 230 ml of ethyl acetate and 110 ml of water. Theresulting mixture was allowed to separate into two layers. The organiclayer so obtained was washed successively with a saturated aqueoussolution of sodium bicarbonate, a 5% aqueous solution of sodium sulfiteand a saturated aqueous solution of sodium chloride, dried overanhydrous magnesium sulfate and then filtered. The filtrate wasconcentrated, followed by the purification of the residue bychromatography on a silica gel column (chloroform:ethyl acetate=10:1→5:1) and then by reversed phase chromatography on a silylizedsilica gel column (CH₃ CN:H₂ O=85:15→90:10). The purified product waslyophilized, whereby 134 mg of the title compound were obtained as whitepowder (yield: 29.0%).

α!_(D) =-74° (c=0.37, MeOH)

MS(FD): M⁺ =1032

EXAMPLE 11 Synthesis of cyclo-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂(Code: PF1022-207)

a. 2-Hydroxy-L-hexanoic acid (abbreviation: H-L-norLEUA-OH, namely, CH₃--(CH₂)₃ --CH(OH)COOH)

In a liquid mixture of 140 ml of 1N--HCl and 10 ml of 1,4-dioxane, 9.18g (70 mmol) of L-norleucine were dissolved. An aqueous solution ofsodium sulfite (14.5 g/20 ml) was added dropwise to the resultingsolution, followed by stirring at room temperature for 3 hours. Afterice cooling, the resultant reaction mixture was added to a liquidmixture of 200 ml of a saturated aqueous solution of sodium chloride and400 ml of ethyl acetate. The resulting mixture was allowed to separateinto two layers. The water layer so obtained was extracted twice with100 ml portions of ethyl acetate. The combined organic layers were driedover anhydrous magnesium sulfate. The magnesium sulfate was filtered offand the filtrate was concentrated under reduced pressure. Theconcentrate obtained was fed as such for use in the next reaction.

b. Benzhydryl 2-hydroxy-L-hexanoate (H-L-norLEUA-OBH)

In 90 ml of ethyl acetate, the 2-hydroxy-L-hexanoic acid(H-L-norLEUA-OH) was dissolved. A solution (15.1 g/30 ml) ofdiphenyldiazomethane in ethyl acetate was added dropwise to theresulting solution, followed by stirring overnight at room temperature.The reaction mixture was adjusted to pH 2 with 2N--HCl, followed bywashing thrice with 200 ml portions of a saturated aqueous solution ofsodium bicarbonate. The organic layer so obtained was dried overanhydrous magnesium sulfate and then filtered. The filtrate wasthereafter concentrated under reduced pressure. The residue obtained wasthen purified by subjecting to chromatography on a silica gel column(toluene:ethyl acetate=20:1), whereby 11.35 g of the title compound wereobtained as pale yellow crystals (yield: 54.7%).

¹ H-NMR(CDCl₃): δ=0.85(t,3H,J=7.0,Me), 1.28-1.83(m,6H,β-,γ-,δ-CH₂),2.70(d,1H,J=6.2,OH), 4.30(dd,1H,J=2.9,6.2,α-H), 6.95(s,1H,CHPh₂),7.25-7.38(m,10H,Ph)

c. Boc-L-MeLeu-D-norLEUA-OBH

In 30 ml of THF, 3.98 g of triphenylphosphine and 5.37 g ofH-L-norLEUA-OBH were dissolved. To the resulting solution, a solution of3.68 g of Boc-L-MeLeu-OH and 2.36 ml of DEAD in 10 ml of THF was addeddropwise, followed by stirring overnight at room temperature. From thereaction solution obtained by this condensation reaction, theprecipitate was filtered off. Ethyl acetate (200 ml) was added to thefiltrate. The resulting mixture was washed successively with a saturatedaqueous solution of sodium bicarbonate, a saturated aqueous solution ofsodium chloride and water, dried over anhydrous magnesium sulfate andfiltered. The filtrate was concentrated. The residue obtained waspurified by subjecting to chromatography on a silica gel column(toluene:ethyl acetate=20:1→10:1), whereby 7.5 g of the title compoundwere obtained as an oil (yield: 95%).

¹ H-NMR(CDCl₃): δ=0.82-0.94(m,9H,δ-Me(MeLeu), ε-Me(norLEUA)),1.44(d,9H,t-Bu), 2.71(d,3H,NMe), 4.74-4.78, 4.98-5.02(each m,1H,α-H(MeLeu)), 5.11(t,1H,α-H(norLEAU)), 6.96(s,1H,CHPh₂),7.26-7.37(m,10H,Ph)

α!_(D) =-9.4° (c=0.55,CHCl₃)

d. Boc-L-MeLeu-D-norLEUA-OH

In a liquid mixture of 50 ml of methanol and 5 ml of water, 5.00 g (9.52mmol) of Boc-L-MeLeu-D-norLEUA-OBH were dissolved. To the resultingsolution, 500 mg of 10% Pd--C were added under a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 2 hours. The catalyst was filtered off withaid of Hyflo Super Cel and the filtrate was concentrated. The residueobtained was fed as such for use in the next reaction.

d. H-L-MeLeu-D-PhLac-OBn

In 2 ml of dichloromethane, 2.41 g (5.0 mmol) of Boc-L-MeLeu-D-PhLac-OBnwere dissolved. To the resulting solution, 2 ml of TFA were added underice cooling, followed by stirring at room temperature for one hour. Thereaction mixture was added with a small quantity of toluene and thenconcentrated. Ethyl acetate (50 ml) was added to the concentrate. Theresulting mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate and water, dried over anhydrous magnesiumsulfate and filtered. The filtrate was then concentrated and the residueobtained was fed as such for use in the next reaction.

e. Boc-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn

In 30 ml of THF, 3.0 g of Boc-L-MeLeu-D-norLEUA-OH, 2.19 g ofH-L-MeLeu-D-PhLac-OBn and 924 mg of HOBt were dissolved. To theresulting solution, 1.41 g of DCC were added under ice cooling, followedby stirring overnight at room temperature. After the precipitate wasfiltered off, the filtrate was concentrated. The residue was added with100 ml of ethyl acetate. The resulting mixture was washed successivelywith 100 ml of a 5% aqueous solution of sodium sulfite, 100 ml of asaturated aqueous solution of sodium bicarbonate and 100 ml of asaturated aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then filtered. The filtrate was concentrated. Theresidue obtained was then purified by subjecting it to chromatography ona silica gel column (toluene:ethyl acetate=10:1), whereby 2.17 g of thetitle compound were obtained as a colorless oil (yield: 52.5%).

¹ H-NMR(CDCl₃): δ=0.83-0.98(m,15H,δ-Me(MeLeu), ε-Me(norLEAU),1.44(d,t-Bu), 2.80(m,6H,NMe), 7.13-7.36(m,10H,Ph)

f. H-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn

In 2 ml of dichloromethane, 985 mg (1.36 mmol) ofBoc-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn were dissolved. TFA (2 ml) wasadded to the resulting solution under ice cooling, followed by stirringat room temperature for 30 minutes. The reaction mixture was added witha small quantity of toluene and then concentrated. Ethyl acetate (30 ml)was added to the concentrate. The resulting mixture was washedsuccessively with a saturated aqueous solution of sodium bicarbonate andwater, dried over anhydrous magnesium sulfate and then filtered. Thefiltrate was concentrated and the residue obtained was fed as such foruse in the next reaction.

g. Boc-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 1.10 g (1.52mmol) of Boc-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn were dissolved. Theresulting solution was added with 110 mg of 10% Pd--C under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 4 hours. The catalyst was filteredoff with aid of Hyflo Super Cel and the filtrate was concentrated. Theresidue obtained was fed as such for use in the next reaction.

h. Boc-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ -OBn

In 10 ml of THF, 892 mg of Boc-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OH, 800mg of L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn and 220 mg of HOBt weredissolved. The resulting solution was added with 337 mg of DCC under icecooling, followed by stirring overnight at room temperature. Theprecipitate as formed was filtered off and the filtrate wasconcentrated. Ethyl acetate (30 ml) was added to the residue. Theresulting mixture was washed successively with a 5% aqueous solution ofsodium sulfite, a saturated aqueous solution of sodium bicarbonate and asaturated aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then filtered. After the filtrate wasconcentrated, the residue as obtained was then purified by subjecting itto chromatography on a silica gel column (toluene:ethylacetate=10:1→5:1, whereby 920 mg of the title compound were obtained asa colorless oil (yield: 54.5%).

α!_(D) =-50.5° (c=0.3,CHCl₃)

i. H-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ -OBn

In 2 ml of dichloromethane, 920 mg (0.74 mmol) ofBoc-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ -OBn were dissolved. Theresulting solution was added with 1 ml of TFA under ice cooling,followed by stirring at room temperature for one hour. The reactionmixture was added with a small quantity of toluene and thenconcentrated, followed by the addition of 30 ml of ethyl acetate. Theresulting mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate and water, dried over anhydrous magnesiumsulfate and filtered. The filtrate was concentrated and the residueobtained was fed as such for use in the next reaction.

j. H-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ -OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 850 mg ofH-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ -OBn were dissolved. Theresulting solution was added with 10% Pd--C under a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 2 hours. The catalyst was filtered off withaid of Hyflo Super Cel. The filtrate was then concentrated. The residuewas supplied as such for use in the next reaction.

k. Cyclo-(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ (Code: PF1022-207)

In a liquid mixture of 500 ml of THF and 150 ml of DMF were dissolved270 mg of lithium chloride, 4.7 g of potassium chloride, 370 mg ofsodium chloride, 1.06 g of cesium chloride and 1.2 g of EDCI.HCl. Themixture so obtained was added with a solution of 633 mg of(L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)₂ -OH, 426 mg of HOBt and 0.13 ml ofNMM in 80 ml of THF, followed by stirring overnight at room temperature.After the solvents were distilled off, the residue was added with 150 mlof ethyl acetate and 80 ml of water. The resulting mixture was allowedto separate into two layers. The organic layer so obtained was washedsuccessively with a saturated aqueous solution of sodium bicarbonate, a5% aqueous solution of sodium sulfite and a saturated aqueous solutionof sodium chloride, dried over anhydrous magnesium sulfate and thenfiltered. The filtrate was concentrated. The residue so obtained waspurified by chromatography on a silica gel column (chloroform:ethylacetate=10:1→5:1), whereby 274 mg of the title compound were obtained aspale yellowish white powder (yield: 35.8%).

α!_(D) =-57.2° (c=0.1,MeOH)

¹ H-NMR(CDCl₃): δ=0.79-1.04(m,30H,δ-Me(MeLeu), ε-Me(norLEAU)),1.37-1.70(m,β-,γ-,δ-CH₂ (norLEUA),β-CH₂, γ-H(MeLeu)),2.72-3.20(m,12H,NMe), 4.88(d,4H,CH₂ --Ph), 5.09-5.92(m,8H,α-H),7.27-7.31(m,10H,Ph)

MS(FD): M⁺ =1032

EXAMPLE 12 Synthesis ofcyclo-(L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac)(Code: PF1022-225)

a.Boc-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn

In 20 ml of methylene chloride, 1.02 g ofBoc-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-OH and 0.98 g ofH-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn were dissolved. The resultingsolution was added with 0.74 ml of diisopropylethylamine and 0.52 g ofBOP-Cl under ice cooling, followed by stirring at the same temperaturefor 16 hours (for the condensation). The reaction mixture was added with50 ml of methylene chloride. The resulting mixture was then washedsuccessively with a 5% aqueous solution of potassium bisulfate, a 7%aqueous solution of sodium bicarbonate and a 20% aqueous solution ofsodium chloride, each 50 ml, dried over anhydrous magnesium sulfate andthen concentrated under reduced pressure. The residue was purified bychromatography on a silica gel column (toluene:ethyl acetate=6:1→5:1),whereby 1.52 g of the title compound were obtained as white powder(yield: 80.5%).

α!_(D) ²⁰ : -58.1° (c=0.21, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.70-1.00(m,27H,δ-Me(MeLeu),ε-ME(norLEAU)),1.44(s,9H,t-Bu), 1.15-1.85(m,21H,β-Me(lac),β-CH₂,γ-H(MeLeu),β-CH₂,γCH₂,δ-CH₂ (norLEUA), 2.65-3.30(m,16H,N-Me,β-CH₂(PhLac)), 4.30-5.50(m,8H,α-H(MeLeu),α-H(PhLac),α-H(Lac),α-H(norLEAU)),5.12(d,2H,J=0.89,CH₂ Ph), 7.10-7.40 (m, 15H, Ph)

b. H-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBn

In 6 ml of methylene chloride, 1.48 g ofBoc-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu- D-PhLac-OBnwere dissolved. To the resulting solution, 3 ml of TFA were addeddropwise under ice cooling, followed by reaction at room temperature for30 minutes (for removal of Boc). The reaction mixture was con-centrated, followed by the addition of toluene to and by azeotropicalremoval of TFA. The residue obtained was dissolved in 100 ml of ethylacetate. The resulting solution was washed successively with a 7%aqueous solution of sodium bicarbonate and a 20% aqueous solution ofsodium chloride, each 100 ml, dried over anhydrous magnesium sulfate andthen concentrated under reduced pressure, whereby 1.37 g of the titlecompound were obtained as a colorless oil. This oil was provided withoutpurification for use in the next reaction.

¹ H-NMR(CDCl₃): δ=0.82-0.99(m,27H,δ-Me(MeLeu), ε-Me(norLEUA)),1.20-1.81(m,21H,β-Me(Lac),β-CH₂ (MeLeu), γ-H(MeLeu),β-CH₂,γ-CH₂,δ-CH₂(norLEUA)), 2.73-3.35(m,16H,N-Me,β-CH₂ (PhLac)),5.06-5.55(m,8H,α-H(MeLeu),α-H(PhLac),α-H(Lac), α-H(norLEAU)),5.12(d,2H,J=0.89,CH₂ Ph), 7.18-7.37(m,15H,Ph)

c. H-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OH

In 26 ml of methanol, 1.32 g ofH-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OBnwere dissolved. To the resulting solution, 0.13 g of 10%palladium-carbon and a drop of acetic acid were added under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for one hour.

The catalyst was filtered off and then, the filtrate was concentrated,whereby 1.21 g of the title compound were obtained as white powder. Thecompound was provided without purification for use in the next reaction.

α!_(D) ²⁰ : -22.1° (c=0.21, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.70-1.05(m,27H,δ-Me(MeLeu),ε-ME(norLEAU)),1.15-1.85(m,21H,β-Me(Lac),β-CH₂ (MeLeu),γ-H(MeLeu),β-CH₂,γCH₂,δ-CH₂(norLEUA), 2.40-3.15(m,16H,N-Me,β-CH₂ (PhLac)),5.05-5.70(m,8H,α-H(MeLeu),α-H(PhLac),α-H(Lac),α-H(norLEAU)),7.25(m,10H,Ph)

d.Cyclo-(L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac(Code: PF022-225)

In 165 ml of THF, 1.18 g ofH-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-norLEUA-L-MeLeu-D-PhLac-OH,0.79 g of HOBt and 0.51 ml of NMM were dissolved. The resulting solutionwas added to a mixture of 0.50 g of lithium chloride, 0.68 g of sodiumchloride, 0.87 g of potassium chloride, 1.97 g of cesium chloride, 2.24g of EDCI-HCl, 1060 ml of THF and 307 ml of DMF, followed by stirring atroom temperature for 16 hours (for the ring-closing reaction). Thereaction mixture was concentrated and the residue obtained was dissolvedin 120 ml of ethyl acetate. The resulting solution was washedsuccessively with a 7% aqueous solution of sodium bicarbonate, a 5%aqueous solution of potassium bisulfate and a 20% aqueous solution ofsodium chloride, each 120 ml, dried over anhydrous magnesium sulfate andthen concentrated under reduced pressure. The residue was purified bychromatography on a silica gel column (toluene:ethyl acetate=3:1→2:1),whereby 0.95 g of the title compound was obtained as white powder(yield: 82.0%).

α!_(D) ²⁰ : -70.6° (c=0.23, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.80-1.05(m,27H,δ-Me(MeLeu),ε-Me(norLEAU)),1.23-1.76(m,21H,β-Me(Lac),β-CH₂ (MeLeu), γ-H(MeLeu),β-CH₂,γ-CH₂,δ-CH₂(norLEUA), 2.67-3.15(m,16H,N-Me,β-CH₂ (PhLac)),5.00-5.70(m,8H,α-H(MeLeu),α-H(PhLac),α-H(Lac),α-H

EXAMPLE 13 Synthesis of cyclo-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂ (Code:PF-1022-209)

a. Boc-L-MeLeu-D-VALA-L-MeLeu-D-Lac-OBn

In 35 ml of THF, 2.22 g of Boc-L-MeLeu-D-VALA-OH, 2.22 g ofH-L-MeLeu-D-Lac-OBn and 1.04 g of HOBt were dissolved. To the resultingsolution, 1.59 g of DCC were added under ice cooling, followed bystirring at 5° C. for 47 hours. The precipitate so formed was filteredoff and the filtrate was then concentrated. The residue was dissolved in100 ml of ethyl acetate. The resulting solution was washed successivelywith 100 ml of a 7% aqueous solution of sodium bicarbonate and 100 ml ofa 30% aqueous solution of sodium chloride, dried over anhydrous sodiumsulfate and then concentrated. The residue was purified bychromatography on a silica gel column (toluene:ethyl acetate=10:1→5:1),whereby 2.12 g of the title compound were obtained (yield: 52%).

α!_(D) ²⁰ : -50° (c=0.17, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.87-1.02(m,18H,δ-Me(Me-Leu),γ-ME(VALA)),1.45(m,9H,t-Bu), 1.51(d,3H,β-Me(Lac)),1.41-1.74(m,7H,β-CH₂,γ-H(Me-Leu),β-H(VALA), 2.84(m,3H,NMe),2.97(d,3H,NMe), 5.15(d,2H,CH₂ Ph),5.01-5.23(m,4H,α-H(MeLeu),α-H(VALA),α-H(Lac)), 7.35(s,5H,Ph)

b. Boc-L-MeLeu-D-VALA-L-MeLeu-D-Lac-OH

In 15 ml of methanol, 1.48 g of Boc-L-MeLeu-D-VALA-L-MeLeu-D-Lac-OBnwere dissolved under a nitrogen atmosphere. To the resulting solution,0.15 g of 10% Pd/C was added, followed by catalytic hydrogenation withhydrogen at room temperature under normal pressure for one hour. Thecatalyst was filtered off with aid of Hyflo Super Cel. The filtrate wasconcentrated, whereby the title compound was obtained as a colorlessoil. The compound was provided without purification for use in the nextreaction.

¹ H-NMR(CDCl₃): δ=0.90-0.98(m,18H,δ-Me(MeLeu),γ-Me(VALA),1.45(s,9H,t-Bu), 1.58(d,3H,β-Me(Lac)), 2.81(s,3H,NMe), 3.06(d,3H,NMe),4.91-5.30(m,4H,α-H(MeLeu), α-H(VALA),α-H(Lac))

c. H-L-MeLeu-D-VALA-L-MeLeu-D-Lac-OBn

In 8 ml of methylene chloride, 1.62 g ofBoc-L-MeLeu-D-VALA-L-MeLeu-D-Lac-OBn were dissolved. To the resultingsolution, 2.4 ml of TFA were added dropwise under ice cooling, followedby stirring at room temperature for one hour. The reaction mixture wasconcentrated, followed by adding toluene to the residue and byazeotropically distilling off TFA and toluene. The residue so obtainedwas dissolved in 80 my of ethyl acetate. The resulting solution waswashed with 80 ml of a 7% aqueous solution of sodium bicarbonate and a30% aqueous solution of sodium chloride, dried over anhydrous magnesiumsulfate and then concentrated, whereby the title compound was obtainedas a colorless oil. The compound was provided without purification foruse in the next reaction.

¹ H-NMR(CDCl₃): δ=0.88-0.99(d×2,12H,δ-Me(MeLeu)), 1.02(d,6H,γ-Me(VALA)),2.37(s3H,NMe), 3.01(d,3H,NMe), 5.20(d,2H,CH₂ Ph),5.06-5.34(m,4H,α-H(MeLeu), α-H(VALA),α-H(Lac)), 7.35(s,5H,Ph)

d. Boc-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂ -OBn

In 28 ml of THF were dissolved 1.12 g ofBoc-L-MeLeu-D-VALA-L-MeLeu-D-Lac-OH, 1.20 g ofL-MeLeu-D-VALA-L-MeLeu-D-Lac-OBn, 0.33 g of HOBt and 0.30 ml oftriethylamine. To the resulting solution, 0.55 g of DCC was added underice cooling, followed by stirring at 5° C. for 2 days. After theprecipitate was filtered off, the filtrate was concentrated. The residuewas then dissolved in 75 ml of ethyl acetate. The resulting solution waswashed successively with 75 ml of a 5% aqueous solution of potassiumbisulfate, 75 ml of a 7% aqueous solution of sodium bicarbonate and 75ml of a 30% aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then concentrated. The residue was purified bychromatography on a silica gel column (toluene:ethyl acetate=5:1),whereby 1.40 g of the title compound were obtained as white prismcrystals (yield: 64.0%).

α!_(D) ²⁰ : -63.1° (c=0.2, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.87-0.95(m,24H,δ-Me(MeLeu)), 1.00(d,12H,γ-Me(VALA),1.45(d,9H,t-Bu), 1.44-1.53(d×2,6H,β-Me(Lac)),1.50-1.76(m,12H,β-CH₂,γ-H(MeLeu)), 2.13(m,2H,β-H(VALA)),2.83-3.12(m,12H,NMe), 5.15(d,2H,CH₂ Ph), 5.02-5.33(m,8H,α-H(MeLeu),α-H(VALA),α-H(Lac)), 7.36(s,5H,Ph)

e. H-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂ -OBn

In 8 ml of methylene chloride, 1.30 g ofBoc-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂ -OBn were dissolved. To theresulting solution, 4 ml of TFA were added dropwise under ice cooling,followed by stirring at the same temperature for one hour. The reactionmixture was concentrated, followed by addition of toluene and byazeotropically distilling off TFA and toluene. The residue so obtainedwas dissolved in 65 ml of ethyl acetate. The resulting mixture waswashed with 65 ml of a 7% aqueous solution of sodium bicarbonate and 35ml of a 30% aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then concentrated, whereby the title compound wasobtained as white prism crystals. The compound was provided withoutpurification for use in the next reaction.

¹ H-NMR(CDCl₃): δ=0.87-1.04(m,36H,β-Me(MeLeu),γ-Me(VALA)),1.45(d×2,6H,β-Me(Lac)), 1.70-1.80(m,12H,β-CH₂,γ-H(MeLeu)),2.15(m,2H,β-H(VALA), 2.92-3.16(m,12H,NMe), 5.17(d,2H,CH₂ Ph),5.02-5.37(m,8H,α-H(MeLeu), α-H(VALA),α-H(Lac)), 7.32(s,5H,Ph)

f. H-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂ -OH

In 12 ml of methanol, 1.15 g of H-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂ -OBnwere dissolved under a nitrogen atmosphere. To the resulting solution,0.12 g of 10% Pd/C was added, followed by catalytic hydrogenation withhydrogen at room temperature under normal pressure for 1.5 hours. Thecatalyst was filtered off with aid of Hyflo Super Cel. The filtrate wasconcentrated, whereby the title compound was obtained as a colorlessoil. The compound was provided without purification for use in the nextreaction.

¹ H-NMR(CDCl₃): δ=0.85-1.06(m,36H,δ-Me(MeLeu),γ-Me(VALA)),1.45(d,6H,β-Me(Lac)), 1.71-1.86(m,12H,β-CH₂, γ-H(MeLeu)),2.20(m,2H,β-H(VALA)), 2.51-3.14(m,12H,NMe), 5.16-5.28(m,8H,α-H(MeLeu),α-H(VALA), α-H(Lac))

g. Cyclo-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂

In 140 ml of THF, 1.0 g of H-(L-MeLeu-D-VALA-L-MeLeu-D-Lac)₂, 0.77 g ofHOBt and 0.51 ml of NMM were dissolved. The resulting solution was addedto a solution, which had been prepared separately by dissolving 0.49 gof lithium chloride, 0.67 g of sodium chloride, 0.85 g of potassiumchloride, 1.93 g of cesium chloride and 2.20 g of EDCI.HCl in a liquidmixture of 900 ml of THF and 260 ml of DMF. The resulting admixture wasstirred at room temperature for 23 hours. The reaction mixture obtainedwas concentrated and the residue was dissolved in 100 ml of ethylacetate. The resulting solution was washed successively with 100 ml of a7% aqueous solution of sodium bicarbonate, 100 ml of a 5% aqueoussolution of potassium bisulfate and 100 ml of a 30% aqueous solution ofsodium chloride, dried over anhydrous magnesium sulfate andconcentrated. The residue was purified by chromatography on a silica gelcolumn (toluene:ethyl acetate=3:1), whereby 0.90 g of the title compoundwas obtained (yield: 92%).

α!_(D) ²⁰ : -65.1° (c=0.1, MeOH)

¹ H-NMR(CDCl₃): δ=0.86-1.08(m,36H,δ-Me(MeLeu),γ-Me(VALA)),1.43(d,6H,β-Me(Lac)), 1.60-2.30(m,14H,β-CH₂, γ-H(MeLeu),β-H(VALA)),2.86-3.22(m,12H,NMe), 4.82-5.90(m,8H,α-H(MeLeu), α-H(VALA),α-H(Lac))

EXAMPLE 14 Synthesis ofcyclo-(L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac)(Code: PF1022-216)

a. H-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In 2 ml of dichloromethane, 993 mg (1.45 mmol) ofBoc-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn were dissolved. The resultingsolution was added with 2 ml of TFA under ice cooling, followed bystirring at room temperature for one hour. The reaction mixture wasadded with a small quantity of toluene and concentrated. Ethyl acetate(25 ml) was then added to the concentrate. The resulting mixture waswashed successively with a saturated aqueous solution of sodiumbicarbonate and water, dried over anhydrous magnesium sulfate andfiltered. The filtrate was concentrated and the residue was fed as suchfor use in the next reaction.

b. Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 984 mg (1.45mmol) of Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OBn (wherein OctA representsthe above-described 2-hydroxyoctanoic acid residue) were dissolved. Theresulting mixture was added with 98 mg of 10% Pd--C under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 3 hours. The catalyst was filteredoff with aid of Hyflo Super Cel and the filtrate was concentrated. Theresidue was provided, as such for use in the next reaction.

c. Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In 20 ml of THF, 845 mg of Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OH, 844 mgof H-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn and 235 mg of HOBt weredissolved. The resulting solution was added with 359 mg of DCC under icecooling, followed by stirring at room temperature for 4 hours. After theprecipitate was filtered off, the filtrate was concentrated. Ethylacetate (100 ml) was added to the residue. The resulting mixture waswashed successively with a 5% aqueous solution of sodium sulfite, asaturated aqueous solution of sodium bicarbonate and a saturated aqueoussolution of sodium chloride, dried over anhydrous magnesium sulfate andfiltered. The filtrate was then concentrated. The residue obtained wasthen purified by subjecting it to chromatography on a silica gel column(toluene:ethyl acetate=10:1), whereby 1.14 g of the title compound wereobtained as a colorless oil (yield: 68.4%).

α!_(D) =45.7° (c=0.1,MeOH)

d. H-L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn

In 2 ml of dichloromethane, 1.13 g (0.984 mmol) ofBoc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn weredissolved. The resulting solution was added with 2 ml of TFA under icecooling, followed by stirring at room temperature for 30 minutes. Thereaction mixture was added with a small quantity of toluene and thenconcentrated. Ethyl acetate(30 ml) was added to the concentrate. Theresulting mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate and water, dried over anhydrous magnesiumsulfate and filtered. The filtrate was concentrated and the residue wasfed as such for use in the next reaction.

e. H-L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 1.140 g ofH-L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OBn weredissolved. The resulting solution was added with 114 mg of 10% Pd--Cunder a nitrogen atmosphere, followed by catalytic hydrogenation withhydrogen at room temperature under normal pressure for 2 hours. Thecatalyst was filtered off with aid of Hyflo Super Cel. The filtrate wasthen concentrated. The residue was provided, as such for use in the nextreaction.

f. Cyclo-(L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac)(Code: PF1022-216)

To a liquid mixture of 650 ml of THF and 195 ml of DMF, 417 mg oflithium chloride, 733 mg of potassium chloride, 575 mg of sodiumchloride, 1.66 g of cesium chloride and 1.89 g of EDCI.HCl were added.The resulting mixture was added with a solution, which had been obtainedin advance by dissolving 865 mg ofH-L-MeLeu-D-OctA-L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-OH, 664 mgof HOBt and 0.22 ml of NMM in 100 ml of THF, followed by stirringovernight at room temperature.

After the solvent was distilled off from the reaction mixture, theresidue was added with 400 ml of ethyl acetate and 400 ml of water. Theresulting mixture was allowed to separate into two layers. The organiclayer so obtained was washed successively with a saturated aqueoussolution of sodium bicarbonate, a 5% aqueous solution of sodium sulfiteand a saturated aqueous solution of sodium chloride, dried overanhydrous magnesium sulfate and then filtered. The filtrate wasconcentrated. The concentrate was purified by chromatography on a silicagel column (chloroform:ethyl acetate=10:1→4:1) and then by reversedphase chromatography on a silylized silica gel column (MeCN:H₂ O=9:1),whereby 210 mg of the title compound were obtained as white powder.

α!_(D) =-73° (c=0.17,MeOH)

¹ H-NMR(CDCl₃): δ=0.80-1.05(m,27H,Me(OctA),δ-Me(MeLeu)),1.29-1.37(m,8H,ζ-,ε-,δ-,γ-,CH₂ (OctA)), 1.41(d,6H,β-Me(Lac)),1.68-1.85(m,14H,β-CH₂,γ-H(MeLeu), β-CH₂ (OctA)), 2.74-3.17(m,12H,NMe),4.46-4.52, 5.07-5.71(m,8H,α-H(MeLeu,OctA,Lac)), 7.27(bs,5H,Ph)

MS(FD): M⁺ =942

EXAMPLE 15 Synthesis of cyclo-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ (Code:PF1022-217)

a. Benzhydryl 2-hydroxy-D-octanoate (H-D-OctA-OBH)

In 25.5 ml of ethyl acetate, 1.68 g of 2-hydroxy-D-octanoic acid weredissolved. To the resulting solution, a solution (2.14 g/19 ml) ofdiphenyldiazomethane in ethyl acetate was added dropwise at roomtemperature over one hour, followed by stirring for four hours. Thereaction mixture was added with 0.63 ml of acetic acid,followed bystirring for three hours to decompose the excess ofdiphenyldiazomethane. The reaction solution was then adjusted to pH 6.5with a 7% aqueous solution of NaHCO₃ under ice cooling. The so treatedsolution was allowed to separate into two layers. The organic layer soobtained was washed with water, dried over anhydrous magnesium sulfateand concentrated. The residue was purified by chromatography on a silicagel column (toluene:ethyl acetate=100:1→50:1), whereby 3.27 g of thetitle compound were obtained as white crystals (yield: 95.5%).

α!_(D) ²⁰ : +22.0° (c=0.28, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.86(t,3H,Me), 1.23-1.42(m,8H,ζ-, ε-,δ-,γ-CH₂),1.62-1.86(m,2H,β-CH₂), 2.75(d,1H,OH), 4.30(m,1H,α-H), 6.95(s,1H,CHPh₂),7.33(s,10H,Ph)

b. Boc-L-MeLeu-D-OctA-OBH

In 30 ml of pyridine, 2.46 g of Boc-L-MeLeu-OH were dissolved. Theresulting solution was added with 3.27 g of H-D-OctA-OBH, 1.62 g of HOBtand 2.48 g of DCC under ice cooling, followed by stirring at the sametemperature for 37 hours. After the precipitate was filtered off, thefiltrate was concentrated. The residue was dissolved in 150 ml of ethylacetate. The resulting solution was washed successively with 150 ml of a5% aqueous solution of potassium bisulfate, 150 ml of a 7% aqueoussolution of sodium bicarbonate and 150 ml of a 5% aqueous solution ofsodium chloride, dried over anhydrous magnesium sulfate and thenconcentrated. The residue was purified by chromatography on a silica gelcolumn (toluene:ethyl acetate=100:1→50:1), whereby 4.73 g of the titlecompound were obtained as a colorless oil (yield: 85.4%).

α!_(D) ²⁰ : -8.5° (c=0.84, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.85(t,3H,Me(OctA)), 0.92(dd,6H,δ-Me(MeLeu)),1.23(m,8H,ζ-,ε-,δ-,γ-CH₂ (OctA)), 1.44(d,9H,t-Bu), 2.72(d,3H,NMe),4.74-5.02 (dd×2,1H,α-H(MeLeu)), 5.12(t,1H,OH(OctA)), 6.90(s,1H,CHPh₂),7.32(s,10H,Ph)

c. Boc-L-MeLeu-D-OctA-OH

In 47 ml of methanol, 4.66 g of Boc-L-MeLeu-D-OctA-OBH were dissolvedunder a nitrogen atmosphere. The resulting solution was added with 0.47g of 10% Pd/C and a drop of acetic acid, followed by catalytichydrogenation with hydrogen at room temperature under normal pressurefor one hour. The catalyst was filtered off with aid of Hyflo Super Cel.The filtrate was concentrated, whereby the title compound was obtainedas a colorless oil. The compound was provided without purification foruse in the next reaction.

¹ H-NMR(CDCl₃): δ=0.88(t,3H,Me(OctA)), 0.95(t,6H,δ-Me(MeLeu)),1.28(m,8H,ζ-,ε-,δ-,γ-CH₂ (OctA)), 1.45(d,9H,t-Bu), 2.81(s,3H,NMe),4.80(dd×2,1H,α-H(MeLeu)), 5.01(t,1H,α-H(OctA))

d. Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OBn

In 50 ml of tetrahydrofuran, 3.26 g of Boc-L-MeLeu-D-OctA-OH, 2.81 g ofH-L-MeLeu-D-Lac-OBn and 1.37 g of HOBt were dissolved. The resultingsolution was added with 2.08 g of DCC and 2.7 ml of pyridine under icecooling, followed by stirring at room temperature for 40 hours. Afterthe precipitate was filtered off, the filtrate was concentrated. Theresidue was dissolved in 300 ml of ethyl acetate. The resulting solutionwas washed successively with 300 ml of a 5% aqueous solution ofpotassium bisulfate, 300 ml of a 7% aqueous solution of sodiumbicarbonate and 300 ml of a 20% aqueous solution of sodium chloride,dried over anhydrous magnesium sulfate and concentrated. The residue waspurified by chromatography on a silica gel column (toluene:ethylacetate=10:1), whereby 3.23 g of the title compound were obtained aswhite crystals (yield: 56.8%).

α!_(D) ²⁰ : -37.9° (c=0.27, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.87-1.01(m,15H,Me(OctA),δ-Me(MeLeu),1.28(m,8H,ζ-,ε-,δ-,γ-CH₂ (OctA), 1.45(d,9H,t-Bu), 1.51(d,3H,β-Me(Lac)),1.64-1.79(m,8H,β-CH₂, γ-H(MeLeu), β-CH₂ (OctA)), 2.87(d,3H,NMe),2.95(d,3H,NMe), 5.07-5.34 (m,6H,CH₂ Ph), α-H(MeLeu), α-H(OctA),α-H(Lac), 7.38(s,5H,Ph)

e. Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OH

In 10 ml of methanol, 990 mg of Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OBnwere dissolved under a nitrogen atmosphere. The resulting solution wasadded with 99 mg of 10% Pd/C and a drop of acetic acid, followed bycatalytic hydrogenation with hydrogen at room temperature under normalpressure for one hour. The catalyst was filtered off with aid of HyfloSuper Cel. The filtrate was then concentrated, whereby the titlecompound was obtained as a colorless oil. The compound was providedwithout purification for use in the next reaction.

¹ H-NMR(CDCl₃): δ=0.84-0.92(m,15H,Me(OctA),δ-Me(MeLeu)),1.23(m,8H,ζ-,ε-,δ-,γ-CH₂ (OctA)), 1.41(d,9H,t-Bu), 1.43(d,3H,β-Me(Lac)),1.62-1.72(m,8H,β-CH₂,γ-H(MeLeu),β-CH₂ (OctA)), 2.77(s,3H,NMe),3.97(d,3H,NMe), 4.80-5.32(m,4H,α-H(MeLeu), α-H(OctA),α-H(Lac))

f. H-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OBn

In 1.5 ml of TFA, 962.4 mg of Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OBn weredissolved under ice cooling. The resulting solution was stirred at thesame temperature for 30 minutes, followed by further stirring at roomtemperature for 30 minutes. The reaction mixture was concentrated.Toluene was then added to the concentrate, which was then azeotropicallydistilled to remove TFA. The residue obtained was dissolved in 75 ml ofethyl acetate. The resulting solution was washed successively with 75 mlof a 7% aqueous solution of sodium bicarbonate and 75 ml of a 30%aqueous solution of sodium chloride, dried over anhydrous magnesiumsulfate and concentrated, whereby the title compound was obtained as acolorless oil. The compound was provided without purification for use inthe next reaction.

¹ H-NMR(CDCl₃): δ=0.85-0.99(m,15H,Me(OctA),δ-Me(MeLeu)),1.27(m,8H,ζ-,ε-,δ-,γ-CH₂ (OctA)), 1.46(d,6H,β-Me(Lac)),1.66-1.82(m,8H,β-CH₂,γ-H(MeLeu), β-CH₂ (OctA)), 2.39(s,3H,NMe),2.96(s,3H,NMe), 5.07-5.36(m,2H,CH₂ Ph),α-H(MeLeu), α-H(OctA), α-H(Lac)),7.37(s,5H,Ph)

g. Boc-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ -OBn

In 13 ml of THF, 857 mg of Boc-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OH, 819 mgof H-L-MeLeu-D-OctA-L-MeLeu-D-Lac-OBn, 237 mg of HOBt and 0.24 ml ofpyridine were dissolved. The resulting solution was added with 362 mg ofDCC under ice cooling, followed by stirring at room temperature for 16hours. The precipitate was filtered off and the filtrate wasconcentrated. The residue was dissolved in 75 ml of ethyl acetate. Theresulting solution was washed successively with 75 ml of a 5% aqueoussolution of potassium bisulfate, 75 ml of a 7% aqueous solution ofsodium bicarbonate and 75 ml of a 20% aqueous solution of sodiumchloride, dried over anhydrous magnesium sulfate and concentrated. Theresidue was purified by chromatography on a silica gel column(toluene:ethyl acetate=5:1), whereby 875 mg of the title compound wereobtained as white crystals (yield: 53.8%).

α!_(D) ²⁰ : -48.8° (c=0.1, CHCl₃)

¹ H-NMR(CDCl₃): δ=0.85-1.01(m,30H,Me(OctA),δ-Me(MeLeu)),1.28(bs,16H,ζ-,ε-,δ-,γ-CH₂ (OctA)), 1.45(d,9H,t-Bu),1.52(d,6H,β-Me(Lac)), 1.72-1.77(m,16H,β-CH₂,γ-H(MeLeu),β-CH₂ (OctA)),2.83-3.10(s,12H,N-Me), 5.14-5.30(m,10H,CH₂ Ph),α-H(MeLeu),α-H(OctA),α-H(Lac)), 7.36(s,5H,Ph)

h. H-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ -OBn

In 4.4 ml of methylene chloride, 870 mg ofBoc-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ -OBn were dissolved. The resultingsolution was added dropwise with 26 ml of TFA under ice cooling,followed by stirring at room temperature for one hour. The reactionmixture was then concentrated. Toluene was added to the concentrate,which was azeotropically distilled to remove TFA therefrom. The residueso obtained was dissolved in 50 ml of ethyl acetate. The resultingsolution was washed with 50 ml of a 7% aqueous solution of sodiumbicarbonate and 50 ml of a 20% aqueous solution of sodium chloride,dried over anhydrous magnesium sulfate and concentrated, whereby thetitle compound was obtained as a colorless oil. The compound wasprovided without purification for use in the next reaction.

¹ H-NMR(CDCl₃): δ=0.86-1.01(m,30H,Me(OctA),δ-Me(MeLeu)),1.26(d,16H,ζ-,ε-,δ-,γ-CH₂ (OctA)),1.41-1.77(m,22H,β-Me(Lac),β-CH₂,γ-H(MeLeu),β-CH.sub.2 (OctA)),2.93(s,6H,NMe), 3.10(d,6H,NMe), 5.20(d,2H,CH₂ Ph),5.08-5.35(m,8H,α-H(MeLeu),α-H(OctA),α-H(Lac)), 7.35(s,5H,Ph)

i. H-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ -OH

In 8 ml of methanol, 794.0 mg of H-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ -OBnwere dissolved under a nitrogen atmosphere. To the resulting solution,80 mg of 10% Pd/C and a drop of acetic acid were added, followed bycatalytic hydrogenation with hydrogen at room temperature under normalpressure for 1.5 hours. The catalyst was filtered off with aid of HyfloSuper Cel. The filtrate was concentrated, whereby the title compound wasobtained as a colorless oil. The compound was provided withoutpurification for use in the next reaction.

¹ H-NMR(CDCl₃): δ=0.88-1.06(m,30H,Me(OctA),δ-CH₃ (MeLeu)),1.26(bs,16H,ζ-,ε-,δ-,γ-CH₂ (OctA)),1.46(d,6H,β-Me(Lac),1.60-1.95(m,16H,β-CH₂,γ-H(MeLeu), β-CH₂ (OctA)),3.02(t,6H,NMe), 3.08(d,6H,NMe), 4.50-5.44(m,8H,α-H(MeLeu),α-H(OctA),α-H(Lac))

j. Cyclo-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂

In 140 ml of THF, 1.00 g of H-(L-MeLeu-D-OctA-L-MeLeu-D-Lac)₂ -OH, 0.77g of HOBt and 0.51 ml of NMM were dissolved. The resulting solution wasadded to a mixture of 0.49 g of lithium chloride, 0.67 g of sodiumchloride, 0.85 g of potassium chloride, 1.93 g of cesium chloride, 2.20g of EDCI.HCl, 900 ml of THF and 260 ml of DMF, followed by stirring atroom temperature for 23 hours. After the reaction mixture wasconcentrated, the residue was dissolved in 100 ml of ethyl acetate. Theresulting solution was washed successively with 100 ml of a 7% aqueoussolution of sodium bicarbonate, 100 ml of 5% potassium bisulfate and 100ml of a 30% aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and concentrated. The residue was purified bychromatography on a silica gel column (toluene:ethyl acetate=3:1),whereby 0.90 g of the title compound was obtained (yield: 92%).

α!_(D) ²⁰ : -46.30 (c=0.1, MeOH)

¹ H-NMR(CDCl₃): δ=0.84-1.06(m,30H,Me(OctA),δ-Me(MeLeu)),1.29(bs,16H,ζ-,ε-,δ-,γ-CH₂ (OctA)),1.43(d,6H,β-Me(Lac),1.68-1.85(m,16H,β-CH₂,γ-H(MeLeu), β-CH₂ (OctA)),2.87(d,6H,NMe), 3.08(d,6H,NMe), 5.41-5.53(m,8H,α-H(MeLeu),α-H(OctA),α-H(Lac))

EXAMPLE 16 Synthesis of cyclo-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ (Code:PF1022-218)

a. Boc-L-Leu-D-Lac-OBH

In 30 ml of THF, 5.13 g (20 mmol) of H-L-Lac-OBH and 10.5 g oftriphenylphosphine were dissolved. To the resulting solution, a solutionof 5.64 g of Boc-L-Leu-OH and 6.08 ml of DEAD in THF were added dropwiseunder ice cooling, followed by stirring at room temperature for 3 days.The reaction mixture was then concentrated. The precipitate was filteredoff, followed by further concentration. Ethyl acetate (300 ml) was addedto the residue. The resulting mixture was washed successively with asaturated aqueous solution of sodium bicarbonate, a saturated aqueoussolution of sodium chloride and water, dried over anhydrous magnesiumsulfate and filtered. The filtrate was then concentrated. The residueobtained was purified by subjecting it to chromatography on a silica gelcolumn (toluene:ethyl acetate=20:1→10:1), whereby 9.57 g of the titlecompound were obtained in a stoichiometric yield (9.57 g).

¹ H-NMR: δ=1.5(s,9H,t-Bu), 4.4(m,1H,CH), 4.9(d,1H,J=6.3,CH),5.2(d,2H,J=4.9,CH₂ Ph), 7.26-7.30(m,10H,Ph)

b. Boc-L-Leu-D-Lac-OH

In a liquid mixture of 100 ml of methanol and 10 ml of water, 9.56 g(20.4 mmol) of Boc-L-Leu-D-Lac-OH were dissolved, followed by theaddition of 956 mg of 10% Pd--C under a nitrogen atmosphere. Theresulting mixture was subjected to catalytic hydrogenation withhydrogen: at room temperature under normal pressure for 2 hours. Thecatalyst was filtered off with aid of Hyflo Super Cel, and the filtratewas concentrated. The residue was provided, as such for use in the nextreaction.

c. Boc-L-Leu-D-Lac-OBn

In 35 ml of DMSO, 5.0 g of Boc-L-Leu-D-Lac-OH were dissolved. To theresulting solution, 1.52 ml of benzyl bromide were added at 35° C. Afterthe temperature was increased to 40° C., the resulting mixture was addedwith 2.94 g of potassium carbonate, followed by stirring at the sametemperature for 5 hours. Ethyl acetate (200 ml) was added to thereaction mixture. The resulting mixture was washed successively withwater and a 10% aqueous solution of sodium chloride, dried overanhydrous magnesium sulfate and filtered. The filtrate was thenconcentrated. The residue as obtained was purified by subjecting it tochromatography on a silica gel column, whereby 4.19 g of the titlecompound were obtained as a colorless oil (yield: 100%).

d. Boc-L-Leu-D-Lac-OH

In 4 ml of dichloromethane, 4.0 g (10.2 mmol) of Boc-L-Leu-D-Lac-OBnwere dissolved. The resulting solution was added with 6 ml of TFA underice cooling, followed by stirring at room temperature for 3 hours. Asmall quantity of toluene was added to the reaction mixture, followed byconcentration. Ethyl acetate (200 ml) was added to the residue obtained.The resulting mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate and water, dried over anhydrous magnesiumsulfate and filtered. The filtrate was then concentrated. The residuewas provided, as such for use in the next reaction.

e. Boc-L-MeLeu-D-PhLac-OH

In a liquid mixture of 60 ml of methanol and 6 ml of water, 5.84 g (12mmol) of Boc-L-MeLeu-D-PhLac-OBn were dissolved. To the resultingsolution, 584 mg of 10% Pd--C were added under a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 4 hours. The catalyst was filtered off withaid of Hyflo Super Cel. The filtrate was concentrated. The residue wasprovided, as such for use in the next reaction.

f. Boc-L-MeLeu-D-PhLac-L-Leu-D-Lac-OBn

In 50 ml of THF, 4.45 g of Boc-L-MeLeu-D-PhLac-OH, 2.87 g ofH-L-Leu-D-Lac-OBn and 1.95 g of HOBt were dissolved. To the resultingsolution, 2.97 g of DCC were added under ice cooling, followed bystirring at room temperature for 3 hours. After the precipitate wasfiltered off, the filtrate was concentrated. Ethyl acetate (100 ml) wasadded to the concentrate. The resulting mixture was washed successivelywith water, a saturated aqueous solution of sodium bicarbonate and asaturated aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and filtered. The filtrate was then concentrated. Theresidue obtained was purified by subjecting it to chromatography on asilica gel column (toluene:ethyl acetate=50:1→10:1), whereby 6.12 g ofthe title compound were obtained as a colorless oil (yield: 90%).

¹ H-NMR: δ=0.85-0.91(m,12H,Me×4), 1.45(s,9H,t-Bu), 2.8(s,3H,NMe),5.07-5.18(m,2H,CH₂ Ph), 7.19-7.39(m,10H,Ph)

g. H-L-MeLeu-D-PhLac-L-Leu-D-Lac-OBn

In 3 ml of dichloromethane, 2.68 g (1.74 mmol) ofBoc-L-MeLeu-D-PhLac-L-Leu-D-Lac-OBn were dissolved. To the resultingsolution, 3 ml of TFA were added under ice cooling, followed by stirringat room temperature for 3 hours. A small quantity of toluene was addedto the reaction mixture, followed by concentration. Ethyl acetate (50ml) was added to the residue as obtained. The resulting mixture waswashed with a saturated aqueous solution of sodium bicarbonate, driedover anhydrous magnesium sulfate and filtered. The filtrate was thenconcentrated. The residue was provided, as such for use in the nextreaction.

h. Boc-L-MeLeu-D-PhLac-L-Leu-D-Lac-OH

In a liquid mixture of 30 ml of methanol and 3 ml of water, 2.68 g (4mmol) of Boc-L-MeLeu-D-PhLac-L-Leu-D-Lac-OBn were dissolved. Theresulting solution was added with 268 mg of 10% Pd--C under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 3 hours. The catalyst was filteredoff with aid of Hyflo Super Cel. The residue was provided, as such foruse in the next reaction.

i. Boc-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ -OBn

In 30 ml of THF, 2.20 g of Boc-L-MeLeu-D-PhLac-L-Leu-D-Lac-OH, 2.18 g ofH-L-MeLeu-D-PhLac-L-Leu-D-Lac-OBn and 649 mg of HOBt were dissolved. Theresulting solution was added with 990 mg of DCC under ice cooling,followed by stirring at room temperature for 4 hours. The precipitatewas filtered off and the filtrate was concentrated. Ethyl acetate (100ml) was added to the concentrate. The resulting mixture was washedsuccessively with a 5% solution of sodium bisulfate, a saturated aqueoussolution of sodium bicarbonate and a saturated aqueous solution ofsodium chloride, dried over anhydrous magnesium sulfate and filtered.The filtrate was concentrated. Ethyl acetate (100 ml) was added to theconcentrate. The resulting mixture was washed successively with a 5%solution of sodium bisulfate, a saturated aqueous solution of sodiumbicarbonate and a saturated aqueous solution of sodium chloride, driedover anhydrous magnesium sulfate and filtered. The filtrate wasconcentrated. The residue as obtained was purified by chromatography ona silica gel column (toluene:ethyl acetate=10:1), whereby 3.18 g of thetitle compound were obtained as a colorless oil (yield: 70.4%).

j. H-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ -OBn

In 6 ml of dichloromethane, 3.10 g (2.75 mmol) ofBoc-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ -OBn were dissolved. To the resultingsolution, 5 ml of TFA were added under ice cooling, followed by stirringat room temperature for one hour. A small quantity of toluene was addedto the reaction mixture, followed by concentration. Ethyl acetate (100ml) was added to the residue obtained. The resulting mixture was washedsuccessively with a saturated aqueous solution of sodium bicarbonate andwater, dried over anhydrous magnesium sulfate and filtered. The filtratewas then concentrated. The residue was provided, as such for use in thenext reaction.

k. H-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ -OH

In a liquid mixture of 30 ml of methanol and 3 ml of water, 3.06 g ofH-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ -OBn were dissolved. The resultingsolution was added with 306 mg of 10% Pd--C under a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 3 hours. The catalyst was filtered off withaid of Hyflo Super Cel, and the filtrate was concentrated. The residueobtained was provided as such for use in the next reaction.

l. Cyclo-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ (code: PF1022-218)

To a liquid mixture of 1875 ml of THF and 563 ml of DMF, 1.16 g oflithium chloride, 2.05 g of potassium chloride, 1.6 g of sodiumchloride, 4.62 g of cesium chloride and 5.262 g of EDCI.HCl were added.The resulting mixture was added at room temperature with a solution,which had been prepared separately by dissolving 2.47 g ofH-(L-MeLeu-D-PhLac-L-Leu-D-Lac)₂ -OH, 1.85 g of HOBt and 0.6 ml of NMMin 300 ml of THF. The admixture obtained was stirred overnight at roomtemperature. After the solvents were distilled off, the residue wasadded with 400 ml of ethyl acetate and 400 ml of water. The resultingmixture was allowed to separate into two layers. The organic layer soobtained was washed successively with a saturated aqueous solution ofsodium bicarbonate, a 5% aqueous solution of sodium sulfite and asaturated aqueous solution of sodium chloride, dried over anhydrousmagnesium sulfate and then filtered. The filtrate was concentrated, andthe residue obtained was purified by chromatography on a silica gelcolumn (chloroform:ethyl acetate=10:1), whereby 1.57 mg of the titlecompound were obtained as colorless powder (yield: 62%).

α!_(D) =-41° (c=0.5,MeOH)

¹ H-NMR(CD₃ OD): δ=0.83-0.94(m,24H,δ-Me(MeLeu,Leu),1.39-1.42(bd,6H,Me(Lac)), 1.69-2.22(m,12H,β-CH₂,γ-H(MeLeu,Leu)),2.93-3.34(m,10H,N-Me(MeLeu), CH₂ (PhLac), 3.74-3.77, 4.52-4.58,4.80-4.85, 5.10-5.31(m,8H,α-H(MeLeu,Leu,PhLac,Lac)), 7.21-7.36(m,10H,Ph)

MS(FD): M⁺ =920

EXAMPLE 17 Synthesis of cyclo-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ (Code:PF1022-219)

a. Boc-L-Leu-D-PhLac-OBn

In 30 ml of pyridine, 2.33 g of Boc-L-Leu-OH, 2.30 g of H-D-PhLac-OBnand 1.34 g of HOBt were dissolved. To the resulting solution, 2.23 g ofDCC were added under ice cooling,and the mixture was stirred overnight.The precipitate was filtered off and the filtrate was added with 400 mlof ethyl acetate. The resulting mixture was washed with a 5% aqueoussolution of potassium bisulfate, a saturated aqueous solution of sodiumbicarbonate and a 5% aqueous solution of sodium chloride, dried overanhydrous magnesium sulfate and filtered. The filtrate was thenconcentrated. The residue obtained was then purified by subjecting tochromatography on a silica gel column (toluene:ethyl acetate=10:1),whereby 3.57 g of the title compound were obtained as an oil (yield:84.5%).

¹ H-NMR(CDCl₃):δ=0.69(d,3H,J=6.5,Me), 0.78(d,3H,J=6.5,Me),1.43(s,9H,t-Bu), 2.35(s,1H,NH), 3.1-3.25(m,1H,CH), 4.36, 4.81(m,1H,CH),5.14(s,2H,CH₂ Ph), 7.13-7.40(m,10H,Ph)

b. H-L-Leu-D-PhLac-OBn

In 2 ml of dichloromethane, 3.14 g (6.7 mmol) of Boc-L-Leu-D-PhLac-OBnwere dissolved. The resulting solution was added with 4 ml of TFA underice cooling, followed by stirring at room temperature for 2 hours. Asmall quantity of toluene was added to the reaction mixture, followed byconcentration. Ethyl acetate (50 ml) was added to the resultant residue.The resulting mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate and water, dried over anhydrous magnesiumsulfate and filtered. The filtrate was then concentrated. The residuewas provided, as such for use in the next reaction.

c. Boc-L-MeLeu-D-Lac-OH

In a liquid mixture of 30 ml of methanol and 3 ml of water, 2.85 g (7mmol) of Boc-L-MeLeu-D-Lac-OBn were dissolved. To the resultingsolution, 285 mg of 10% Pd--C were added under a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 15 hours. The catalyst was filtered off withaid of Hyflo Super Cel. The filtrate was concentrated. The residue wasprovided, as such for use in the next reaction.

d. Boc-L-MeLeu-D-Lac-L-Leu-D-PhLac-OBn

In 30 ml of THF, 2.20 g of Boc-L-MeLeu-D-Lac-OH, 2.21 g ofH-Leu-L-D-PhLac-OBn and 948 mg of HOBt were dissolved. The resultingsolution was added with 1.73 g of DCC under ice cooling, followed bystirring at room temperature for 3 hours. After the precipitate wasfiltered off, the filtrate was concentrated. Ethyl acetate (100 ml) wasadded to the residue as obtained. The resulting mixture was washedsuccessively with water, a saturated aqueous solution of sodiumbicarbonate and a saturated aqueous solution of sodium chloride, driedover anhydrous magnesium sulfate and filtered. The filtrate was thenconcentrated. The residue obtained was then purified by subjecting it tochromatography on a silica gel column (toluene:ethyl acetate=10:1→5:1),whereby 6.38 g of the title compound were obtained as a colorless oil(yield: 72.2%).

e. H-L-MeLeu-D-Lac-L-Leu-D-PhLac-OBn

In 3 ml of dichloromethane, 1.55 g (2.31 mmol) ofBoc-L-MeLeu-D-Lac-L-Leu-D-PhLac-OBn were dissolved. To the resultingsolution, 3 ml of TFA were added under ice cooling, followed by stirringat room temperature for 3 hours. A small quantity of toluene was addedto the reaction mixture, followed by concentration. Ethyl acetate (50ml) was added to the residue obtained. The resulting mixture was washedwith a saturated aqueous solution of sodium bicarbonate, dried overanhydrous magnesium sulfate and filtered. The filtrate was thenconcentrated. The residue was fed to as such for use in the nextreaction.

f. Boc-L-MeLeu-D-Lac-L-Leu-D-PhLac-OH

In a liquid mixture of 15 ml of methanol and 1.5 ml of water, 1.61 g(2.4 mmol) of Boc-L-MeLeu-D-Lac-L-Leu-D-PhLac-OBn were dissolved. Theresulting solution was added with 160 mg of 10% Pd--C under a nitrogenatmosphere, followed by catalytic hydrogenation with hydrogen at roomtemperature under normal pressure for 3 hours. The catalyst was filteredoff with aid of Hyflo Super Cel. The residue was fed to as such for usein the next reaction.

g. Boc-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ -OBn

In 20 ml of THF, 1.25 g of Boc-L-MeLeu-D-Lac-L-Leu-D-PhLac-OH, 1.29 g ofL-MeLeu-D-Lac-L-Leu-D-PhLac-OBn and 373 mg of HOBt were dissolved. Theresulting solution was added with 569 mg of DCC under ice cooling,followed by stirring at room temperature for 4 hours. After theprecipitate was filtered off, the filtrate was concentrated. Ethylacetate (100 ml) was added to the residue. The resulting mixture waswashed successively with a 5% aqueous solution of sodium sulfite, asaturated aqueous solution of sodium bicarbonate and a saturated aqueoussolution of sodium chloride, dried over anhydrous magnesium sulfate andfiltered. The filtrate was then concentrated. The residue obtained waspurified by subjecting it to chromatography on a silica gel column(toluene:ethyl acetate=3:1), whereby 1.29 g of the title compound wereobtained as a colorless oil (yield: 49.7%).

h. H-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ -OBn

In 4 ml of dichloromethane, 1.28 g (1.13 mmol) ofBoc-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ -OBn were dissolved. To the resultingsolution, 2 ml of TFA were added under ice cooling, followed by stirringat room temperature for one hour. A small quantity of toluene was addedto the reaction mixture, followed by concentration. Ethyl acetate (100ml) was added to the residue obtained. The resulting mixture was washedsuccessively with a saturated aqueous solution of sodium bicarbonate andwater, dried over anhydrous magnesium sulfate and filtered. The filtratewas then concentrated. The residue was supplied as such for use in thenext reaction.

i. H-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ -OH

In a liquid mixture of 10 ml of methanol and 1 ml of water, 1.22 g ofH-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ -OBn were dissolved. To the resultingsolution, 122 mg of 10% Pd--C were added under a nitrogen atmosphere,followed by catalytic hydrogenation with hydrogen at room temperatureunder normal pressure for 3 hours. The catalyst was filtered off withaid of Hyflo Super Cel. The filtrate was concentrated. The residue wassupplied as such for use in the next reaction.

j. Cyclo-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ (Code: PF 1022-219)

To a liquid mixture of 750 ml of THF and 225 ml of DMF were added 446 mgof lithium chloride, 786 mg of potassium chloride, 615 mg of sodiumchloride, 1.7 g of cesium chloride and 2.02 g of EDCI.HCl. The resultingmixture was added with a solution, which had been prepared by dissolving990 mg of H-(L-MeLeu-D-Lac-L-Leu-D-PhLac)₂ -OH, 712 mg of HOBt and 0.23ml of NMM in 120 ml of THF. The admixture so obtained was stirredovernight at room temperature. After the solvents were distilled offtherefrom, the resultant residue was added with 200 ml of ethyl acetateand 100 ml of water. The resulting mixture was allowed to separate intotwo layers. The resulting organic layer was washed successively with asaturated aqueous solution of sodium bicarbonate, a 5% aqueous solutionof sodium sulfite and a saturated aqueous solution of sodium chloride,dried over anhydrous magnesium sulfate and then filtered. The filtratewas concentrated. The residue obtained was then purified by subjectingit to chromatography on a silica gel column (chloroform:ethylacetate=10:1), whereby 586 mg of the title compound were obtained aswhite powder (yield: 55.8%).

α!_(D) =-94° (c=0.48, MeOH)

¹ H-NMR(CD₃ OD): δ=0.86-0.99(m,24H,δ-Me(MeLeu,Leu),1.34-1.37(bd,6H,Me(Lac)), 1.70-2.27(m,12H,β-CH₂,γ-H(MeLeu)),3.18-3.31(m,10H,N-Me(MeLeu), CH₂ (PhLac), 4.02-4.08, 4.55-4.62,4.80-4.95, 5.45-5.55(m,8H,α-H(MeLeu,Leu,PhLac,Lac)),7.12-7.22(,m,10H,Ph)

MS(FD): M⁺ =920

Illustrated in the following Examples 18-34 are processes for preparingthe PF 1022 derivatives by introduction of substituent(s).

EXAMPLE 18 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-t-Bu)-L-MeLeu-D-Lac)(code: PF 1022-215)

In methylene chloride (10 ml) placed in a tube, 701 mg ofcyclo(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA-L-MeLeu-D-Lac)(namely, PF 1022E substance) were dissolved, followed by the addition of1.4 ml of isobutene and 0.11 ml of concentrated sulfuric acid at -40° C.After tube sealing, the temperature of the resulting mixture was allowedto rise back to room temperature. The resulting mixture was stirred for2 hours. The reaction mixture was ice-cooled and then adjusted to pH 9.0with 0.6 ml of triethylamine, followed by concentration. The residue wasdissolved in 70 ml of ethyl acetate, followed by washing successivelywith 70 ml of a 5% aqueous solution of potassium bisulfate and 70 ml ofa 30% aqueous solution of sodium chloride, drying over anhydrousmagnesium sulfate and concentration. The residue was purified bychromatography on a silica gel column (chloroform:ethyl acetate=4:1),whereby 578 mg of the title compound were obtained (yield: 77.6%).

α!_(D) ²⁰ : -92.0° (c=0.1, MeOH)

¹ H-NMR(CDCl₃): δ=0.80-1.05(m,24H,δ-Me(MeLeu)), 1.32(d,9H,t-Bu),1.40(d,6H,β-Me(Lac)), 1.10-1.80(m,12H, β-CH₂,γ-H(MeLeu),2.70-3.20(m,16H,NMe,β-CH₂ (PhLac)),5.30-5.80(m,8H,α-H(MeLeu),α-H(PhLac),α-H(Lac)), 7.00(dd,4H,t-BuOC₆ H₄),7.26(d,5H,Ph)

EXAMPLE 19 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OCOC₁₇H₃₅)-L-MeLeu-D-Lac) (code: PF-1022-006)

In 1.8 ml of THF, 202 mg of the PF 1022E substance, 105 mg of stearicacid, 59.2 mg of HOBt and 0.05 ml of NMM were dissolved, followed by theaddition of 82.3 mg of EDCI.HCl under ice cooling. The resulting mixturewas stirred at 4° C. for 24 hours. The reaction mixture was diluted with40 ml of ethyl acetate and 20 ml of hexane, followed by washingsuccessively with 40 ml of water, 40 ml of a saturated aqueous solutionof sodium bicarbonate and 40 ml of a saturated aqueous solution ofsodium chloride, filtration using a small quantity of silica gel forchromatography, and drying over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure. The resulting whitecrystalline powder was purified by chromatography on a silica gel column(chloroform:hexane=1:1→chloroform:ethyl acetate=5:1), followed bycrystallization from hexane-methanol-water, whereby 173 mg of the titlecompound were obtained as white crystals (m.p. 47°-48° C., yield:67.1%).

α!_(D) ²² : -71° (c=0.15, MeOH)

¹ H-NMR(CD³ OD): δ=0.79-1.28(m,60H,C₁₆ H₃₃ CH₂),1.25-1.88(12H,β-CH₂,γH(MeLeu)), 1.38(d×2,3H,β-Me(Lac)),2.27-2.56(m,2H,α-H,C₁₆ H₃₃ CH₂), 3.05-3.21(m,4H,β-CH₂ (PhLac,TYRA),2.82-3.04(m,12H,NMe), 4.77-5.81(m,8H,α-H), 7.04-7.35(m,9H,aromatic)

MS(FAB): (M+H)⁺ =1231

EXAMPLE 20 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(3,5-diiode)-L-MeLeu-D-Lac)(Code: PF1022-011)

To a solution of 203 mg of the PF 1022E substance in 5 ml of methylenechloride, 130 mg of sodium acetate were added. The resulting mixture wasice-cooled, followed by the addition of 210 mg of iodine. After icecooling for 30 minutes, the temperature of the reaction mixture wasallowed to rise back to room temperature. Triethylamine (0.06 ml) wasadded to the reaction mixture, followed by stirring at the sametemperature for 2.5 hours. The reaction mixture was added with 3 ml of a10% aqueous solution of sodium thiosulfate, followed by the addition of20 ml of water and 30 ml of chloroform. The resulting mixture wasallowed to separate into two layers. The water layer was extracted witha liquid mixture of 20 ml of ethyl acetate and 8 ml of hexane. Thecombined organic layers were filtered using a small quantity of silicagel for chromatography, followed by drying over anhydrous sodiumsulfate. The solvents were then distilled off under reduced pressure.The residue so obtained was purified by chromatography on a silica gelcolumn (chloroform:ethyl acetate=5:1), whereby 109 mg of the titlecompound were obtained as crystals (m.p. 124-126° C., yield: 42.4%).

α!_(D) ²⁰ : -92° (c=0.08, MeOH)

¹ H-NMR(CD₃ OD): δ=0.78-1.08(m,27H,γ-Me(MeLeu),β-Me(Lac)),1.33-1.91(m,12H,β-CH₂,γ-H(MeLeu)), 1.39(d×2,3H,β-Me(Lac)),2.82-3.04(m,12H,NMe), 2.91-3.21(m,4H,β-CH₂ (PhLac,TYRA),4.77-5.82(m,8H,α-H), 7.24-7.70(m,7H,aromatic)

MS(FAB): (M+H)⁺ =1217

EXAMPLE 21 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-Me,3,5-di-I)-L-MeLeu-D-Lac)(Code: PF 1022-012)

In 2 ml of THF, 70 mg of the 3,5-diiodide of the PF 1022E substance weredissolved, followed by the addition of 0.02 ml of methyl iodide and 7 mgof 60% sodium hydride under ice cooling. The resulting mixture wasstirred at 0° C. for 3.5 hours. The reaction mixture was diluted with 20ml of ethyl acetate, followed by washing with 10 ml of a saturatedaqueous solution of sodium chloride, filtering through a small quantityof silica gel for chromatography, and drying over anhydrous sodiumsulfate. The solvent was then distilled off under reduced pressure. Theresidue so obtained was purified by preparative chromatography on asilica gel column (chloroform:ethyl acetate=3:1) and then, bychromatography on a silica gel column (chloroform:ethyl acetate=4:1),whereby 43.8 mg of the title compound were obtained as white powder(m.p. 108°-110° C., yield: 61.9%).

α!_(D) ²⁰ : -98° (c=0.1, MeOH)

¹ H-NMR(CD₃ OD): δ=0.79-1.08(m,27H,γ-Me(MeLeu),β-Me(Lac)),1.12-1.90(m,12H,β-CH₂,γ-H(MeLeu)), 1.38, 1.39(d×2,3H,β-Me(Lac)),2.81-3.14(m,12H,NMe), 4.76-5.82(m,8H,α-H), 7.24-7.81(m,7H,aromatic)

MS(FAB): (M+H)⁺ =1231

EXAMPLE 22 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OCOO-isoBu)-L-MeLeu-D-Lac)(Code: PF 1022-013)

In a liquid mixture of 5 ml of ethyl ether and 5 ml of methylenechloride, 263 mg of Cbz-GABA (namely, γ-aminobutyric acid) weredissolved. To the resulting solution, 0.3 ml of triethylamine and 0.15ml of isobutyl chloroformate were added under ice cooling. Five minutesafter the addition, 640 mg of PF 1022E were added to the resultingmixture, which was then stirred for one hour under ice-cooling. Thereaction mixture was diluted with ethyl acetate, followed by washingonce with 50 ml of a 5% aqueous solution of potassium bisulfate andtwice with 50 ml portions of a saturated aqueous solution of sodiumbicarbonate, filtering through a small quantity of silica gel forchromatography, and drying over anhydrous sodium sulfate. The solventwas then distilled off under reduced pressure. The residue so obtainedwas purified by chromatography on a silica gel column (chloroform:ethylacetate=20:1→10:1→8:1), followed by crystallization from ether-hexane,whereby 414 mg of the title compound were obtained as prism crystals(m.p. 95-97° C., yield: 58.6%).

¹ H-NMR(CDCl₃): δ=0.80-1.04(m,27H,γ-Me(MeLeu),β-Me(Lac)),1.00(d,6H,J=6.7,γMe(isobutyl)), 1.40(d×2,3H,β-Me(Lac)),1.42-1.79(m,12H,β-CH₂,γ-H(MeLeu)), 2.05(m,1H,β-H(isobutyl)),2.72-3.06(m,12H,NMe), 3.05-3.18(m,4H,β-CH₂ (PhLac,TYRA),4.02(d×2,2H,α-CH₂ (isobutyl)), 4.48-5.71(m,8H,α-H),7.08-7.14,7.21-7.40(m,9H,aromatic)

MS(FAB): (M+H)⁺ =1065

EXAMPLE 23 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OEt)-L-MeLeu-D-Lac)(Code: PF 1022-016)

In 4 ml of THF, 153 mg of the PF 1022E substance were dissolved,followed by the addition of 0.02 ml of ethyl iodide and 15 mg of 60%sodium hydride under ice cooling. The resulting mixture was stirred at0° C. for 30 minutes. The reaction mixture was diluted with 30 ml ofethyl acetate, followed by washing with a liquid mixture of a 20%aqueous solution of sodium chloride and 10 ml of a 10% aqueous solutionof sodium thiosulfate, filtering through a small quantity of silica gelfor chromatography, and drying over anhydrous sodium sulfate. Thesolvent was thereafter distilled off under reduced pressure. The residueso obtained was purified by chromatography on a silica gel column(chloroform: ethyl acetate=8:1→5:1), followed by lyophilization using1,4-dioxane, whereby 158 mg of the title compound were obtained.

α!_(D) ²⁰ : -67° (c=0.11, CHCl₃)

¹ H-NMR(CD₃ OD): δ=0.78-1.05(m,27H,δ-Me(MeLeu),δ-Me(Lac)),1.37(t,3H,Me(ethyl)), 1.38(d×2,3H,β-Me(Lac)),1.46-1.90(m,12H,β-CH₂,γ-H(MeLeu)), 2.81-3.00(m,12H,NMe),3.01-3.20(m,4H,β-CH₂ (PhLac,TYRA)), 4.00(q,2H,CH₂ (ethyl)),4.76-5.81(m,8H,α-H), 6.85-7.33(m,9H,aromatic)

MS(FAB): (M+H)⁺ =993

EXAMPLE 24 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-n-Pr)-L-MeLeu-D-Lac)(Code: PF 1022-018)

In 8 ml of THF, 217 mg of the PF 1022E substance were dissolved,followed by the addition of 0.2 ml of n-propyl iodide and 27 mg of 60%sodium hydride under ice cooling. The resulting mixture was stirred at0° C. for 2 hours and then at room temperature for 4 hours. The reactionmixture was diluted with 50 ml of ethyl acetate, followed by washingwith 30 ml of a saturated aqueous solution of sodium chloride, filteringthrough a small quantity of silica gel for chromatography and dryingover anhydrous sodium sulfate. The solvent was then distilled off underreduced pressure. The residue so obtained was purified by chromatographyon a silica gel column (chloroform:ethyl acetate=7:1→3:1), followed bylyophilization using 1,4-dioxane, whereby 107 mg of the title compoundwere obtained (yield: 47%).

¹ H-NMR(CD₃ OD): δ=0.78-1.11(m,30H,δ-Me(MeLeu),β-Me(Lac), OCH₂ CH₂ CH₃),1.30-1.89(m,14H,β-CH₂,γ-H(MeLeu), OCH₂ CH₂ CH₃), 1.38,1.39(d×2,J=7.0,3H,β- Me(Lac)), 2.82-3.00(m,12H,NMe),2.92-3.21(m,4H,β-CH₂ (PhLac,TYRA), 3.90, 3.97(each t,2H,OCH₂ CH₂ CH₃),4.75-5.83(m,8H,α-H), 6.85-7.33(m,9H,aromatic)

MS(FAB): (M+H)⁺ =1007

EXAMPLE 25 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-iso-Pr)-L-MeLeu-D-Lac)(Code: PF 1022-019)

In 8 ml of THF, 206 mg of the PF 1022E substance were dissolved,followed by the addition of 0.2 ml of isopropyl iodide and 19 mg of 60%sodium hydride under ice cooling. The resulting mixture was stirred at0° C. for 2 hours and then at room temperature for 4 hours. The reactionmixture was diluted with 50 ml of ethyl acetate, followed by washingsuccessively with 12 ml of a saturated aqueous solution of sodiumchloride and 28 ml of a 5% aqueous solution of sodium thiosulfate,filtering through a small quantity of silica gel for chromatography, anddrying over anhydrous sodium sulfate. The solvent was distilled offunder reduced pressure. The residue so obtained was purified bychromatography on a silica gel column (chloroform:ethylacetate=8:1→4:1), followed by lyophilization using 1,4-dioxane, whereby108 mg of the title compound were obtained (yield: 50%).

¹ H-NMR(CD₃ OD): δ=0.78-1.06(m,27H,γ-Me(MeLeu),β-Me(Lac), 1.29,1.30(d×2,6H,Me(isopropyl), 1.38, 1.39(d×2,3H,β-Me(Lac)),1.45-1.89(m,12H,β-CH₂,γ-H(MeLeu)), 2.82-3.00(m,12H,NMe),2.91-3.21(m,4H,β-CH₂ (PhLac,TYRA), 4.55(m,1H,CH(iso-propyl)),4.74-5.82(m,8H,α-H), 6.84-7.34(m,9H,aromatic)

MS(FAB): (M+H)⁺ =1007

EXAMPLE 26 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-All)-L-MeLeu-D-Lac)(Code: PF 1022-020)

In 8 ml of THF, 206 mg of the PF 1022E substance were dissolved,followed by the addition of 0.18 ml of allyl iodide and 20 mg of 60%sodium hydride under ice cooling. The resulting mixture was stirred at0°C. for one hour. The reaction mixture was diluted with 50 ml of ethylacetate, followed by washing with 30 ml of a saturated aqueous solutionof sodium chloride, filtering through a small quantity of silica gel forchromatography, and drying over anhydrous sodium sulfate. The solventwas distilled off under reduced pressure. The residue so obtained waspurified by chromatography on a silica gel column (chloroform:ethylacetate=7:1→4:1), followed by crystallization from ether-hexane, whereby158 mg of the title compound were obtained (yield: 73.8%).

¹ H-NMR(CD₃ OD): δ=0.78-1.07(m,27H,γ-Me(MeLeu),β-Me(Lac), Me(Lac),1.30-1.90(m,12H,β-CH₂,γ-H(MeLeu)), 1.38, 1.39(d×2,3H,β-Me(Lac)),2.81-3.00(m,12H,NMe), 2.90-3.20(m,4H,β-CH₂ (PhLac,TYRA),4.52(m,2H,allyl), 4.74-5.17, 5.44-5.82(m,8H,α-H), 5.20-5.25, 5.34-5.41,6.05(each m,each 1H,allyl), 6.88-7.35(m,9H,aromatic)

MS(FAB): (M+H)⁺⁼ 1005

EXAMPLE 27 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-n-Bu)-L-MeLeu-D-Lac)(Code: PF 1022-021)

In 8 ml of THF, 204 mg of the PF 1022E substance were dissolved,followed by the addition of 0.24 ml of n-butyl iodide and 16 mg of 60%sodium hydride under ice cooling. The resulting mixture was stirred at0° C. for one hour and then at room temperature for one hour. Thereaction mixture was diluted with 50 ml of ethyl acetate, followed bywashing with 30 ml of a saturated aqueous solution of sodium chloride,filtering through a small quantity of silica gel for chromatography, andthen drying over anhydrous sodium sulfate. The solvent was thendistilled off under reduced pressure. The residue so obtained waspurified by chromatography on a silica gel column (chloroform:ethylacetate=8:1→4:1), followed by lyophilization using 1,4-dioxane, whereby159 mg of the title compound were obtained (yield: 73.7%).

¹ H-NMR(CD₃ OD): δ=0.77-1.05(m,30H,δ-Me(MeLeu),β-Me(Lac), Me(butyl)),1.28-1.90(m,16H,β-CH2,γ-H(MeLeu), OCH₂ CH₂ CH₂ CH₃), 1.38,1.39(d×2,3H,β-Me(Lac)), 2.82-3.00(m,12H,NMe),2.92-3.20(m,4H,β-CH2PhLac,TYRA), 3.94(m,1H,OCH₂ CH₂ CH₂ CH₃),4.75-5.81(m,8H,α-H), 6.85-7.33(m,9H,aromatic)

MS(FAB): (M+H)⁺ =1021

EXAMPLE 28 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(OBn)-L-MeLeu-D-Lac)(Code: PF 1022-022)

In 9 ml of THF, 305 mg of the PF 1022E substance were dissolved,followed by the addition of 0.07 ml of benzyl iodide and 28 mg of 60%sodium hydride under ice cooling. The resulting mixture was stirred at0° C. for 1.5 hours. The reaction mixture was diluted with 50 ml ofethyl acetate, followed by washing with 40 ml of a saturated aqueoussolution of sodium chloride, filtering through a small quantity ofsilica gel for chromatography, and drying over anhydrous sodium sulfate.The solvent was then distilled off under reduced pressure. The residueso obtained was purified by chromatography on a silica gel column(chloroform:ethyl acetate=10:1), followed by lyophilization using aliquid mixture of 1,4-dioxane and water, whereby 320 mg of the titlecompound were obtained (yield: 95.8%).

¹ H-NMR(CD₃ OD): δ=0.78-1.06(m,27H,δ-Me(MeLeu),β-Me(Lac)),1.25-1.90(m,12H,β-CH₂,γ-H(MeLeu)), 1.38, 1.39(d×2,3H,β-Me(Lac)),2.82-3.00(m,12H,NMe), 2.86-3.20(m,4H,β-CH₂ (PhLac,TYRA)), 4.74-4.78,5.17-5.82(m,8H,α-H), 5.05(s,2H,CH₂ Ph), 6.95-7.50(m,14H,aromatic)

MS(FAB): (M+H)⁺ =1055

EXAMPLE 29 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(3,5-di-Cl)-L-MeLeu-D-Lac)(Code: PF 1022-023)

In 15 ml of methylene chloride, 301 mg of the PF 1022E substance weredissolved, followed by the addition of 0.22 ml of t-butyl hypochloriteunder ice cooling. The resulting mixture was stirred at the sametemperature for 40 minutes. The reaction mixture was added with 30 ml ofa 5% aqueous solution of sodium thiosulfate and 30 ml of methylenechloride. The resulting mixture was allowed to separate into two layers.The organic layer so obtained was filtered through a small quantity ofsilica gel for chromatography, followed by drying over anhydrous sodiumsulfate. The solvent was then distilled off under reduced pressure. Theresidue so obtained was purified by chromatography on a silica gelcolumn (chloroform:ethyl acetate=8:1→4:1), whereby 120 mg of the titlecompound were obtained (yield: 37.2%).

¹ H-NMR(CD₃ OD): δ=0.78-1.07(m,27H,δ-Me(MeLeu),β-Me(Lac)),1.26-1.90(m,12H,β-CH₂,γ-H(MeLeu)), 1.40,1.41(d×2,3H,β-Me(Lac)),2.80-3.18(m,12H,NMe), 2.80-3.20(m,4H,β-CH₂ (PhLac,TYRA)),4.78-5.85(m,8H,α-H), 7.24-7.35(m,7H,aroma-tic)

MS(FAB): (M+H)⁺ =1033, 1035

EXAMPLE 30 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(3,5-di-Br)-L-MeLeu-D-Lac)(Code: PF 1022-025)

In 20 ml of methylene chloride, 406 mg of the PF 1022E substance weredissolved. The resulting solution was added with 0.12 ml oftriethylamine and 0.07 ml of bromine under ice cooling, followed bystirring at 0° C. for 20 minutes. The reaction mixture was added with 40me of chloroform and 30 ml of a 5% aqueous solution of sodiumthiosulfate. The resulting mixture was allowed to separate into twolayers. The water layer so obtained was extracted with a liquid mixtureof 20 ml of ethyl acetate and 10 ml of hexane. The combined organiclayers were filtered through a small quantity of silica gel forchromatography, followed by drying over anhydrous sodium sulfate. Thesolvent was then distilled off under reduced pressure. The residue soobtained was purified by chromatography on a silica gel column(chloroform:ethyl acetate=9:1→7:1), whereby 420 mg of the title compoundwere obtained (yield: 89.0%).

α!: -90° (c=0.2, MeOH)

¹ H-NMR(CD₃ OD): δ=0.78-1.07(m,27H,δ-Me(MeLeu),β-Me(Lac)),1.38(d,J=6.7,β-Me(Lac)), 1.28-1.91(m,12H,β-CH₂,γ-H(Me- Leu)),2.82-3.01(m,12H,NMe), 2.92-3.21(m,4H,β-CH₂ (PhLac,TYRA),4.77-5.82(m,8H,α-H), 7.26-7.46(m,7H,aromatic)

MS(FAB):(M+H)⁺⁼ 1121, 1123, 1125

EXAMPLE 31 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(3,5-di-Br,O-Me)-L-MeLeu-D-Lac)(Code: PF 1022-026)

In 8 ml of THF, 200 mg of the 3,5-dibromide of the PF 1022E substancewere dissolved. The resulting solution was added with 0.1 ml of methyliodide and 16 mg of 60% sodium hydride under ice cooling, followed bystirring at 0° C. for 1.5 hours. The reaction mixture was diluted with50 ml of ethyl acetate, followed by washing with 30 ml of a saturatedaqueous solution of sodium chloride, filtering through a small quantityof silica gel for chromatography, and drying over anhydrous sodiumsulfate. The solvents were then distilled off under reduced pressure.The residue so obtained was purified by chromatography on a silica gelcolumn (chloroform:ethyl acetate=10:1), whereby 108 mg of the titlecompound were obtained (yield: 53.1%).

¹ H-NMR(CD₃ OD): δ=0.78-1.08(m,27H,δ-Me(MeLeu),β-Me(Lac)),1.28-1.90(m,12H,δ-Me(MeLeu),β-Me(Lac)),1.28-1.90(m,12H,β-CH₂,γ-H(MeLeu)), 1.38, 1.39(d×2,J=6.7,β-Me(Lac)),2.81-3.13(m,12H,NMe), 2.90-3.20(m,4H,β-CH₂ (PhLac,TYRA)),4.77-5.82(m,8H,α-H), 7.24-7.59(m,7H,aromatic)

MS(FAB):(M+H)⁺ =1135, 1137, 1139

EXAMPLE 32 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-Oct)-L-MeLeu-D-Lac)(Code: PF 1022-029)

In 5 ml of THF, 253 mg of the PF 1022E substance were dissolved. Theresulting solution was added with 2.4 ml of octyl iodide and 23 mg of60% sodium hydride under ice cooling, followed by stirring at 0° C. forone hour. The reaction mixture was diluted with 40 ml of ethyl acetateand 10 ml of hexane, followed by washing with 30 ml of a saturatedaqueous solution of sodium chloride, filtering through a small quantityof silica gel for chromatography and drying over anhydrous sodiumsulfate. The solvents were then distilled off under reduced pressure.The residue so obtained was purified by chromatography on a silica gelcolumn (chloroform:ethyl acetate=20:1→10:1→5:1), whereby 199 mg of thetitle compound were obtained (yield: 70.6%).

EXAMPLE 33 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-THP)-L-MeLeu-D-Lac)(Code: PF 1022-224)

In 5 ml of methylene chloride, 202 mg of the PF 1022E substance weredissolved. The resulting solution was added with 4.5 mg ofp-toluenesulfonic acid hydrate and 0.04 ml of 2,3-dihydropyran, followedby stirring at room temperature for 40 minutes. The reaction mixture wasadded with 0.01 ml of triethylamine, followed by concentration. Theresidue so obtained was purified by chromatography on a silica gelcolumn (ethyl acetate:hexane=1:1), whereby 200 mg of the title compoundwere obtained (yield: 91%).

¹ H-NMR(CDCl3): δ=0.79-1.05(m,27H,δ-Me(MeLeu),β-Me(Lac)),1.23-2.05(m,18H,β-CH₂,γ-H(MeLeu), OCH₂ (CH₂)₃ (THP)),1.40(d,J=6.7,β-Me(Lac)), 2.72-3.01(s×10,12H,NMe), 3.02-3.18(m,4H,β-CH₂(PhLac,TYRA), 3.57-3.62, 3.85-3.91(m,each 1H,OCH₂ CH₂ (THP)),4.47-5.70(m,9H,α-H,OCHO(THP)), 6.94-6.99, 7.11-7.15,7.21-7.31(m,9H,aromatic)

MS(FAB):(M+H)⁺ =1049

EXAMPLE 34 Preparation ofcyclo-(L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-TYRA(O-Tr)-L-MeLeu-D-Lac)(Code: PF 1022-223)

In 16 ml of methylene chloride, 410 mg of the PF 1022E substance weredissolved. The resulting solu- tion was added with 210 mg of tritylchloride, 0.08 ml of triethylamine and 16 mg of 4-dimethylaminopyridine,followed by stirring at room temperature for 24 hours. The reactionmixture was diluted with 8 ml of toluene, followed by purification bychromatography on a silica gel column (ethyl acetate:hexane=2:1),whereby 287 mg of the title compound were obtained as white powder(yield: 56%).

m.p. 109°-115° C. (dec.)

¹ H-NMR(CD₃ OD): δ=0.78-1.05(m,27H,δ-Me(MeLeu),β-Me(Lac)),1.24-1.80(m,12H,β-CH₂,γ-H(MeLeu)), 1.40(d,3H,J=7,β-Me(Lac)),2.51-3.01(s×10,12H,NMe), 2.77-3.18(m,4H,β-CH₂ (PhLac,TYRA)),4.41-5.70(m,8H,α-H), 6.57-6.62, 6.81-6.84, 7.18-7.31,7.41-7.45(m,24H,aromatic)

MS(FAB):(M+H)⁺ =1207

INDUSTRIAL FIELD OF UTILIZATION OF INVENTION

The PF 1022 derivatives represented by the general formula (I) which areprovided herein by the present invention each has anthelminticactivities against various parasitic worms which are parasitic on humanbodies, domestic animals and companion animals. They are thereforeuseful as anthelmintic agent for prevention or treatment of parasiticinfections.

We claim:
 1. A cyclodepsipeptide represented by the following generalformula: ##STR37## wherein R^(2a) and R^(4a) are each a cyclohexylmethylgroup or benzyl group, provided that at least one of R^(2a) and R^(4a)is a cyclohexylmethyl group.
 2. A cyclodepsipeptide represented by thefollowing general formula: ##STR38## wherein R^(1b), R^(2b), R^(3b) andR^(4b) are each a cyclohexylmethyl group or benzyl group, provided thatat least one of R^(1b), R^(2b), R^(3b) and R^(4b) is a cyclohexylmethylgroup.
 3. A cyclodepsipeptide represented by the following generalformula: ##STR39## wherein R^(1e), R^(2e) and R^(3e) are each a linearor branched alkyl group containing 1 to 11 carbon atoms, and may be thesame or different from each other, and G, L and M denote independently ahydrogen, a halo group, hydroxyl group, an alkoxy group, a loweralkenyloxy group, a phenyl-lower alkoxy group, an alkylcarbonyloxygroup, tetrahydropyranyloxy group or trityloxy group.
 4. Acyclodepsipeptide represented by the following general formula:##STR40## wherein G', L' and M' denote independently a substituentchosen from a halo group, hydroxyl group, alkoxy group, lower alkenyloxygroup, phenyl-lower alkoxy group, alkylcarbonyloxy group,tetrahydropyranyloxy group or trityloxy group.
 5. A cyclodepsipeptiderepresented by the following general formula: ##STR41## wherein G', L'and M' denote independently a substituent chosen from a halo group,hydroxyl group, alkoxy group, lower alkenyloxy group, phenyl-lower alkylgroup, alkylcarbonyloxy group, tetrahydropyranyloxy group or trityloxygroup.
 6. A cyclodepsipeptide, PF 1022E substance represented by thefollowing formula: ##STR42##
 7. An anthelmintic composition,characterized in that the composition comprises, as the activeingredient, a cyclodepsipeptide which is the hydrogenated derivative ofthe PF 1022 substance represented by the formula (I-i-a) as defined inclaim
 1. 8. An anthelmintic composition, characterized in that thecomposition comprises, as the active ingredient, a cyclodepsipeptidewhich is the hydrogenated derivative of the PF 1022 B substancerepresented by the formula (I-i-b) as defined in claim
 2. 9. Ananthelmintic composition, characterized in that the compositioncomprises, as the active ingredient, a cyclodepsipeptide represented bythe formula (I-iv) as defined in claim
 3. 10. An anthelminticcomposition, characterized in that the composition comprises, as theactive ingredient, a cyclodepsipeptide represented by the formula(I-vi-a) as defined in claim
 4. 11. An anthelmintic composition,characterized in that the composition comprises, as the activeingredient, a cyclodepsipeptide represented by the formula (I-vi-b) asdefined in claim
 5. 12. An anthelmintic composition, characterized inthat the composition comprises, as the active ingredient, acyclodepsipeptide which is the hydrogenated derivative of the PF 1022Esubstance as defined in claim 6.